Transgenic animals

ABSTRACT

The present invention provides improved methods and compositions for the generation of transgenic non-human animals. The present invention permits the introduction of exogenous nucleic acid sequences into the genome of unfertilized eggs (e.g., pre-maturation oocytes and pre-fertilization oocytes) by microinjection of infectious retrovirus into the perivitelline space of the egg. The methods of the present invention provide an increased efficiency of production of transgenic animals with a reduced rate of generating animals which are mosaic for the presence of the transgene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/821,984 filed Mar. 20, 1997 now U.S. Pat. No. 6,080,912.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to improved methods for the generation oftransgenic non-human animals. In particular, the present inventionrelates to the introduction of retroviral particles into theperivitelline space of gametes, zygotes and early stage embryos to allowthe insertion of genetic material into the genome of the recipientgamete or embryo.

BACKGROUND OF THE INVENTION

The ability to alter the genetic make-up of animals, such asdomesticated mammals such as cows, pigs and sheep, allows a number ofcommercial applications. These applications include the production ofanimals which express large quantities of exogenous proteins in aneasily harvested form (e.g., expression into the milk), the productionof animals which are resistant to infection by specific microorganismsand the production of animals having enhanced growth rates orreproductive performance. Animals which contain exogenous DNA sequencesin their genome are referred to as transgenic animals.

The most widely used method for the production of transgenic animals isthe microinjection of DNA into the pronuclei of fertilized embryos. Thismethod is efficient for the production of transgenic mice but is muchless efficient for the production of transgenic animals using largemammals such as cows and sheep. For example, it has been reported that1,000 to 2,000 bovine embryos at the pronuclear stage must bemicroinjected to produce a single transgenic cow at an estimated cost ofmore than $500,000 (Wall et al., J. Cell. Biochem. 49:113 [1992]).Furthermore, microinjection of pronuclei is more difficult when embryosfrom domestic livestock (e.g., cattle, sheep, pigs) is employed as thepronuclei are often obscured by yolk material. While techniques for thevisualization of the pronuclei are known (i.e., centrifugation of theembryo to sediment the yolk), the injection of pronuclei is an invasivetechnique which requires a high degree of operator skill.

Alternative methods for the production include the infection of embryoswith retroviruses or with retroviral vectors. Infection of both pre- andpost-implantation mouse embryos with either wild-type or recombinantretroviruses has been reported (Janenich, Proc. Natl. Acad. Sci. USA73:1260 [1976]; Janenich et al., Cell 24:519 [1981]; Stuhlmann et al.,Proc. Natl. Acad. Sci. USA 81:7151 [1984]; Jahner et al., Proc. Natl.Acad Sci. USA 82:6927 [1985]; Van der Putten et al., Proc. Natl. AcadSci. USA 82:6148-6152 [1985]; Stewart et al., EMBO J. 6:383-388 [1987]).The resulting transgenic animals are typically mosaic for the transgenesince incorporation occurs only in a subset of cells which form thetransgenic animal. The consequences of mosaic incorporation ofretroviral sequences (i.e., the transgene) include lack of transmissionof the transgene to progeny due to failure of the retrovirus tointegrate into the germ line, difficulty in detecting the presence ofviral sequences in the founder mice in those cases where the infectedcell contributes to only a small part of the fetus and difficulty inassessing the effect of the genes carried on the retrovirus.

In addition to the production of mosaic founder animals, infection ofembryos with retrovirus (which is typically performed using embryos atthe 8 cell stage or later) often results in the production of founderanimals containing multiple copies of the retroviral provirus atdifferent positions in the genome which generally will segregate in theoffspring. Infection of early mouse embryos by co-culturing earlyembryos with cells producing retroviruses requires enzymatic treatmentto remove the zona pellucida (Hogan et al., In Manipulating the MouseEmbryo: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., [1994], pp. 251-252). In contrast tomouse embryos, bovine embryos dissociate when removed from the zonapellucida. Therefore, infection protocols which remove the zonapellucida cannot be employed for the production of transgenic cattle orother animals whose embryos dissociate or suffer a significant decreasein viability upon removal of the zona pellucida (e.g., ovine embryos).

An alternative means for infecting embryos with retroviruses is theinjection of virus or virus-producing cells into the blastocoele ofmouse embryos (Jahner, D. et al., Nature 298:623 [1982]). As is the casefor infection of eight cell stage embryos, most of the founders producedby injection into the blastocoele will be mosaic. The introduction oftransgenes into the germline of mice has been reported usingintrauterine retroviral infection of the midgestation mouse embryo(Jahner et al., supra [1982]). This technique suffers from a lowefficiency of generation of transgenic animals and in addition producesanimals which are mosaic for the transgene.

Infection of bovine and ovine embryos with retroviruses or retroviralvectors to create transgenic animals has been reported. These protocolsinvolve the micro-injection of retroviral particles or growth arrested(i.e., mitomycin C-treated) cells which shed retroviral particles intothe perivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990]; and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [19951). PCT International Application WO90/08832 describes the injection of wild-type feline leukemia virus Binto the perivitelline space of sheep embryos at the 2 to 8 cell stage.Fetuses derived from injected embryos were shown to contain multiplesites of integration. The efficiency of producing transgenic sheep waslow (efficiency is defined as the number of transgenics producedcompared to the number of embryos manipulated); only 4.2% of theinjected embryos were found to be transgenic. Haskell and Bowen (supra)describe the micro-injection of mitomycin C-treated cells producingretrovirus into the perivitelline space of 1 to 4 cell bovine embryos.The use of virus-producing cells precludes the delivery of a controlledamount of viral particles per embryo. The resulting fetuses containedbetween 2 and 12 proviruses and were shown to be mosaic for proviralintegration sites, the presence of provirus, or both. The efficiency ofproducing transgenic bovine embryos was low; only 7% of the injectedembryos were found to be transgenic.

The art needs improved methods for the production of transgenic animals,particularly for the production of transgenics using large domesticlivestock animals. The ideal method would be simple to perform and lessinvasive than pronuclear injection, efficient, would produce mosaictransgenic founder animals at a low frequency and would result in theintegration of a defined number of copies of the introduced sequencesinto the genome of the transgenic animal.

SUMMARY OF THE INVENTION

The present invention provides improved methods and compositions for theproduction of transgenic non-human animals. In one embodiment, thepresent invention provides a composition comprising a non-humanunfertilized oocyte comprising a heterologous oligonucleotide (i.e., aheterologous polynucleotide) integrated into the genome of the oocyte.In a preferred embodiment the unfertilized oocyte is a pre-maturationoocyte. In another preferred embodiment the unfertilized oocyte is apre-fertilization oocyte. The present invention is not limited by thenature of the heterologous oligonucleotide contained within the genomeof the oocyte. In a preferred embodiment, the heterologousoligonucleotide is the proviral form of a retroviral vector.

The invention is not limited by the nature of the retroviral vectoremployed. Retroviral vectors containing a variety of genes may beemployed. For example, the retroviral vector may contain sequencesencoding proteins which modify growth rate, size and/or carcasscomposition (e.g., bovine growth hormone or other growth hormones) orforeign proteins of commercial value that are expressed in, andharvested from, a particular tissue component (e.g., blood or milk). Theretroviral vector may contain genes that confer disease resistance toviruses or other microorganisms, including DNA sequences that aretranscribed into RNA sequences that catalytically cleave specific RNAs(i.e., ribozymes) such as viral RNAs and DNA sequences that aretranscribed into anti-sense RNA of an essential gene of a pathogenicmicroorganism. The above protein-encoding genes and DNA sequences areexamples of “genes of interest.”

The compositions of the present invention are not limited by the natureof the non-human animal employed to provide oocytes. In a preferredembodiment, the non-human animal is a mammal (e.g., cows, pigs, sheep,goats, rabbits, rats, mice, etc.). In a particularly preferredembodiment, the non-human animal is a cow.

The present invention further provides a method for introducing aheterologous polynucleotide into the genome of a non-human unfertilizedoocyte, comprising: a) providing: i) a non-human unfertilized eggcomprising an oocyte having a plasma membrane and a zona pellucida, theplasma membrane and the zona pellucida defining a perivitelline space;ii) an aqueous solution comprising a heterologous polynucleotide; and b)introducing the solution comprising the heterologous polynucleotide intothe perivitelline space under conditions which permit the introductionof the heterologous polynucleotide into the genome of the oocyte. Themethod of the present invention is not limited by the nature of theheterologous polynucleotide employed. In a preferred embodiment, theheterologous polynucleotide encodes a protein of interest. In aparticularly preferred embodiment, the heterologous polynucleotide iscontained within genome of a recombinant retrovirus.

The method of the present invention may be practiced using unfertilizedeggs comprising a pre-maturation oocyte. Alternatively, the method ofthe present invention may employ pre-fertilization oocytes as theunfertilized egg.

When a recombinant retrovirus is employed infectious retroviralparticles comprising the heterologous polynucleotide are preferentiallyemployed. The method of the present invention is not limited by thenature of the infectious retrovirus employed to deliver nucleic acidsequences to an oocyte. Any retrovirus which is capable of infecting thespecies of oocyte to be injected may be employed. In a preferredembodiment, the infectious retrovirus comprises a heterologousmembrane-associated protein. In a preferred embodiment, the heterologousmembrane-associated protein is a G glycoprotein selected from a viruswithin the family Rhabdoviridae. In another preferred embodiment, theheterologous membrane associated protein is selected from the groupconsisting of the G glycoprotein of vesicular stomatitis virus, Piryvirus, Chandipura virus, Spring viremia of carp virus and Mokola virus.In a particularly preferred embodiment, the heterologousmembrane-associated protein is the G glycoprotein of vesicularstomatitis virus.

The method of the present invention is not limited by the nature of thenon-human animal employed to provide oocytes. In a preferred embodiment,the non-human animal is a mammal (e.g., cows, pigs, sheep, goats,rabbits, rats, mice, etc.). In a particularly preferred embodiment, thenon-human animal is a cow.

The present invention further provides a method for the production of atransgenic non-human animal comprising: a) providing: i) an unfertilizedegg comprising an oocyte having a plasma membrane and a zona pellucida,the plasma membrane and the zona pellucida defining a perivitellinespace; ii) an aqueous solution containing infectious retrovirus; b)introducing the solution containing infectious retrovirus into theperivitelline space under conditions which permit the infection of theoocyte; and c) contacting the infected oocyte with sperm underconditions which permit the fertilization of the infected oocyte toproduce an embryo. In a preferred embodiment, the method of the presentinvention further comprises, following the fertilization of the infectedoocyte, the step of transferring the embryo into a hormonallysynchronized non-human recipient animal (i.e., a female animalhormonally synchronized to stimulate early pregnancy). In anotherpreferred embodiment, the method comprises the step of allowing thetransferred embryo to develop to term. In still another referredembodiment, at least one transgenic offspring is identified from theoffspring allowed to develop to term.

The method of the present invention may be practiced using unfertilizedeggs comprising a pre-maturation oocyte. Alteratively, the method of thepresent invention may employ pre-fertilization oocytes as theunfertilized egg.

When pre-maturation oocytes are employed in the method of the presentinvention, the method may further comprise, following the introductionof the solution containing infectious retrovirus into the pre-maturationoocyte, the further step of culturing the infected pre-maturation oocyteunder conditions which permit the maturation of the pre-maturationoocyte. The art is well aware of culture conditions which permit the invitro maturation of pre-maturation oocytes from a variety of mammalianspecies.

The method of the present invention is not limited by the nature of theinfectious retrovirus employed to deliver nucleic acid sequences to anoocyte. Any retrovirus which is capable of infecting the species ofoocyte to be injected may be employed. In a preferred embodiment, theinfectious retrovirus comprises a heterologous membrane-associatedprotein. In a preferred embodiment, the heterologous membrane-associatedprotein is a G glycoprotein selected from a virus within the familyRhabdoviridae. In another preferred embodiment, the heterologousmembrane-associated protein is selected from the group consisting of theG glycoprotein of vesicular stomatitis virus, Piry virus, Chandipuravirus, Spring viremia of carp virus and Mokola virus. In a particularlypreferred embodiment, the heterologous membrane-associated protein isthe G glycoprotein of vesicular stomatitis virus.

The method of the present invention is not limited by the nature of thenon-human animal employed to provide oocytes. In a preferred embodiment,the non-human animal is a mammal (e.g., cows, pigs, sheep, goats,rabbits, rats, mice, etc.). In a particularly preferred embodiment, thenon-human animal is a bovine.

The present invention further provides compositions comprising a stablymaintained recombinant mammalian zygote, wherein the zygote comprises apolynucleotide containing the proviral form of a retroviral vectorintegrated into the genome of the zygote. In particularly preferredembodiments, the mammalian zygote is a bovine zygote, while in otherpreferred embodiments, the zygote is any mammalian zygote. Indeed, it isnot intended that the present invention be limited to any particularanimal species. In still other embodiments, the proviral form of theretroviral vector encodes a protein of interest. In yet furtherpreferred embodiments, the recombinant retroviral vector comprisesMoloney murine leukemia virus LTR. However, it is not intended that thepresent invention be limited to any particular retroviral LTR. Indeed,it is contemplated that other retroviral LTRs, including, but notlimited, to mouse mammary tumor virus LTR, will find use in the presentinvention.

The present invention also provides methods for introducing apolynucleotide contained within the genome of a recombinant retrovirusinto the genome of a mammalian zygote, comprising: a) providing: 1) amammalian zygote having a plasma membrane and a zona pellucida, whereinthe plasma membrane and zona pellucida define a perivitelline space; ii)an aqueous solution comprising a polynucleotide contained within thegenome of a recombinant retrovirus; and b) introducing the solutioncomprising the polynucleotide contained within the genome of arecombinant retrovirus into the perivitelline space, under conditionswhich permit the introduction of the polynucleotide contained within thegenome of the recombinant retrovirus into the genome of the zygote, suchthat the polynucleotide is stably maintained. In particularly preferredembodiments of the method, the efficiency of the introduction of thepolynucleotide into the genome of the zygote is at least twenty percent.In still other embodiments, the efficiency ranges from approximatelytwenty percent to one hundred percent. In yet other preferredembodiments, the polynucleotide contained within the genome of therecombinant retrovirus encodes a protein of interest. In furtherembodiments, the method further comprises the step of transferring thezygote into a mammalian female recipient that is hormonally synchronizedto simulate early pregnancy, thereby giving a transferred embryo. Inother particularly preferred embodiments, the method further comprisesthe step of allowing the transferred embryo to develop to term. Infurther embodiments, the method comprises the additional step ofidentifying at least one transgenic offspring. In other particularlypreferred embodiments, the present invention provides transgenic animalsproduced according to the above methods. In particularly preferredembodiments, the mammalian zygote is a bovine zygote, while in otherpreferred embodiments, the zygote is any other mammalian zygote. Indeed,is not intended that the present invention be limited to any particularanimal species.

In still other embodiments of the above methods and transgenic animals,the recombinant retrovirus comprises Moloney murine leukemia virus longterminal repeat. However, it is not intended that the present inventionbe limited to any particular retroviral LTR. Indeed, it is contemplatedthat other retroviral LTRs, including, but not limited to mouse mammarytumor virus LTR, will find use in the present invention. In particularlypreferred embodiments, the protein of interest is expressed by thetransgenic offspring. In some embodiments, the protein of interest isexpressed in at least one body fluid of the transgenic offspring. Insome particularly preferred embodiments, the expression of the proteinof interest is preferentially mammary-specific expression.

In further embodiments of the above methods and transgenic animals, therecombinant retrovirus comprises a heterologous membrane-associatedprotein. In some embodiments, the heterologous membrane-associatedprotein is a G glycoprotein selected from a virus within the familyRhabdoviridae. In other embodiments, the G glycoprotein is selected fromthe group comprising the G glycoprotein of vesicular stomatitis virus,Piry virus, Chandipura virus, Spring viremia of carp virus, Rabiesvirus, and Mokola virus.

The present invention also provides methods for producing transgenicnon-human animals, wherein the genome of the transgenic non-human animalcomprises a polynucleotide encoding a recombinant retrovirus and atleast one protein of interest, comprising the steps of: a) providing: i)a non-human mammalian zygote having a plasma membrane and a zonapellucida, wherein the plasma membrane and the zona pellucida define aperivitelline space; ii) an aqueous solution comprising a polynucleotidecontained within the genome of a recombinant retrovirus; b) introducingthe solution comprising the polynucleotide contained within the genomeof a recombinant retrovirus into the perivitelline space underconditions which permit the introduction of the polynucleotide containedwithin the genome of a recombinant retrovirus into the genome of thezygote, such that the polynucleotide is stably maintained in arecombinant zygote; c) transferring the recombinant zygote into anon-human female mammalian recipient that is hormonally synchronized tosimulate early pregnancy, thereby giving a transferred embryo; d)allowing the transferred embryo to develop to term to produce atransgenic animal. In some particularly˜embodiments, at least oneprotein of interest is expressed by the transgenic animal. In otherpreferred embodiments, the recombinant retrovirus comprises Moloneymurine leukemia virus long terminal repeat. However, it is not intendedthat the present invention be limited to any particular retroviral LTR.Indeed, it is contemplated that other retroviral LTRs, including, butnot limited to mouse mammary tumor virus LTR, will find use in thepresent invention.

In still other embodiments of the above methods, the efficiency of theintroduction of the polynucleotide is at least twenty percent. In stillother embodiments, the efficiency ranges from approximately twentypercent to one hundred percent. In further particularly preferredembodiments, the expression of the polynucleotide is preferentiallymammary-specific expression. In other embodiments, the methods comprisethe further step of mating the transgenic animal to a non-transgenicanimal under conditions such that transgenic offspring are produced. Inparticularly preferred embodiments, the transgenic offspring express thepolynucleotide. In other particularly preferred embodiments, theexpression of the polynucleotide is mammary-specific expression. In yetother particularly preferred embodiments, the mammalian zygote is abovine zygote, while in other preferred embodiments, the zygote is anyother mammalian zygote. Indeed, is not intended that the presentinvention be limited to any particular animal species.

The present invention also provides methods for expressing a protein ofinterest, wherein the protein of interest is encoded by a polynucleotidecontained within the genome of a recombinant retrovirus, comprising thesteps of: a) providing: i) a non-human mammalian zygote having a plasmamembrane and a zona pellucida, wherein the plasma membrane and the zonapellucida define a perivitelline space; ii) an aqueous solutioncomprising a polynucleotide encoding a protein of interest containedwithin the genome of a recombinant retrovirus; and b) introducing thesolution comprising the polynucleotide encoding a protein of interestcontained within the genome of a recombinant retrovirus into theperivitelline space, under conditions which permit the introduction ofthe polynucleotide contained within the genome of a recombinantretrovirus into the genome of the zygote, such that the polynucleotideis stably maintained; and c) allowing the zygote to develop into viablenon-human animal, under conditions such that the protein of interest isexpressed by the non-human animal.

In some preferred embodiments of the above methods, the recombinantretrovirus comprises Moloney murine leukemia virus long terminal repeat.However, it is not intended that the present invention be limited to anyparticular retroviral LTR., Indeed, it is contemplated that otherretroviral LTRs, including, but not limited, to mouse mammary tumorvirus LTR, will find use in the present invention. In yet otherpreferred embodiments, introduction of the polynucleotide into thegenome of the zygote is at least twenty percent. In still otherembodiments, the efficiency ranges from approximately twenty percent toone hundred percent. In yet other embodiments, the polynucleotidecontained within the genome of a recombinant retrovirus encodes a viralprotein. In other embodiments, viral protein is hepatitis B surfaceantigen. In still other embodiments, the present invention providesprotein produced according to the above methods. In yet otherembodiments, the method further comprises the step of harvesting theexpressed protein of interest. In further embodiments, the expressedprotein is expressed in the body fluids of the non-human animal. Inparticularly preferred embodiments, body fluids are selected from thegroup consisting of blood, milk, semen, and urine. In particularlypreferred embodiments, the mammalian zygote is a bovine zygote, while inother preferred embodiments, the zygote is any mammalian zygote. Indeed,it is not intended that the present invention be limited to anyparticular animal species.

The present invention also provides methods for expressing a protein ofinterest wherein the protein of interest is encoded by a polynucleotidecontained within the genome of a recombinant retrovirus, and thepolynucleotide is integrated into the genome of a mammalian unfertilizedoocyte, comprising the steps of. a) providing: i) an unfertilizedmammalian egg comprising an oocyte having a plasma membrane and a zonapellucida, wherein the plasma membrane and the zona pellucida define aperivitelline space; ii) an aqueous solution containing recombinantretrovirus, wherein the recombinant retrovirus comprises apolynucleotide encoding a protein of interest; b) introducing thesolution containing recombinant retrovirus into the perivitelline spaceunder conditions which permit the infection of the oocyte to provide aninfected oocyte; c) contacting the infected oocyte with sperm underconditions which permit the fertilization of the infected oocyte toproduce an embryo; d) transferring the embryo into a hormonallysynchronized mammalian recipient animal; e) allowing the embryo todevelop into at least one viable transgenic mammalian animal, underconditions such that the protein of interest is expressed by thetransgenic mammalian animal.

In some preferred embodiments, the unfertilized oocyte is apre-maturation oocyte. In other embodiments, following the introductionof the solution containing infectious retrovirus into the pre-maturationoocyte, the method comprises the further step of culturing the infectedpre-maturation oocyte under conditions which permit the maturation ofthe pre-maturation oocyte. In other preferred embodiments, theunfertilized oocyte is a pre-fertilization oocyte.

In still other preferred embodiments, the method further comprises thestep of identifying at least one transgenic offspring. In particularlypreferred embodiments, the mammal is a bovine. However, it is notintended that the present invention be limited to any particular animalspecies.

In further preferred embodiments, the recombinant retrovirus comprisesMoloney murine leukemia virus long terminal repeat. However, it is notintended that the present invention be limited to any particularretroviral LTR. Indeed, it is contemplated that other retroviral LTRs,including, but not limited, to mouse mammary tumor virus LTR, will finduse in the present invention. In yet other preferred embodiments, theexpression of the protein of interest is preferentially mammary specificexpression. In some particularly preferred embodiments of the method,the introduction of the polynucleotide into the genome of the infectedoocyte, is greater than twenty percent. In still other embodiments, theefficiency ranges from approximately twenty percent to one hundredpercent. In some preferred embodiments, the polynucleotide containedwithin the genome of a recombinant retrovirus encodes a viral protein.In some particularly preferred embodiments the viral protein ishepatitis B surface antigen. In alternative particularly preferredembodiments, the expressed protein is expressed in the body fluids ofthe mammalian animal. In some particularly preferred embodiments, thebody fluids are selected from the group consisting of blood, milk,semen, and urine. In still other embodiments, the methods furthercomprise the step of f) harvesting the expressed protein of interest.The present invention also provides a protein of interest expressedusing the above methods.

In yet other embodiments of the methods, the recombinant retroviruscomprises a heterologous membrane-associated protein. In someembodiments, the heterologous membrane-associated protein is a Gglycoprotein selected from a virus within the family Rhabdoviridae. Inyet other embodiments, the G glycoprotein is selected from the groupcomprising the G glycoprotein of vesicular stomatitis virus, Piry virus,Chandipura virus, Spring viremia of carp virus and Mokola virus.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic showing the production of pre-maturationoocytes, pre-fertilization oocytes and fertilized oocytes (zygotes).

FIG. 2(A-C) shows an autoradiogram of a Southern blot of genomic DNAisolated from the skin (A) and blood (B) of calves derived frompre-fertilization oocytes and zygotes which were injected withpseudotyped LSRNL retrovirus (C).

FIG. 3(A-B) shows an ethidium bromide stained agarose gel containingelectrophoresed PCR products which were amplified using neo gene primers(A) or HBsAg primers (B) from the blood and skin of calves derived frompre-fertilization oocytes and zygotes injected with pseudotyped LSRNLretrovirus.

FIG. 4(A-B) shows an ethidiurn bromide stained agarose gel containingelectrophoresed PCR products amplified using the neo gene primers (A) orHBsAg primers (B) from skin samples obtained from twin calves, who wereoffspring of a transgenic bull.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “egg,” when used in reference to a mammalianegg, means an oocyte surrounded by a zona pellucida and a mass ofcumulus cells (follicle cells) with their associated proteoglycan. Theterm “egg” is used in reference to eggs recovered from antral folliclesin an ovary (these eggs comprise pre-maturation oocytes) as well as toeggs which have been released from an antral follicle (a rupturedfollicle).

As used herein, the term “oocyte” refers to a female gamete cell andincludes primary oocytes, secondary oocytes and mature, unfertilizedovum. An oocyte is a large cell having a large nucleus (i.e., thegerminal vesicle) surrounded by ooplasm. The ooplasm containsnon-nuclear cytoplasmic contents including mRNA, ribosomes,mitochondria, yolk proteins, etc. The membrane of the oocyte is referredto herein as the “plasma membrane.”

The term “pre-maturation oocyte,” as used herein refers to a femalegamete cell following the oogonia stage (i.e., mitotic proliferation hasoccurred) that is isolated from an ovary (e.g., by aspiration) but whichhas not been exposed to maturation medium in vitro. Those of skill inthe art know that the process of aspiration causes oocytes to begin thematuration process but that completion of the maturation process (i.e.,formation of a secondary oocyte which has extruded the first polar body)in vitro requires the exposure of the aspirated oocytes to maturationmedium. Pre-maturation oocytes will generally be arrested at the firstanaphase of meiosis.

The term “pre-fertilization oocyte” as used herein, refers to a femalegamete cell such as a pre-maturation oocyte following exposure tomaturation medium in vitro but prior to exposure to sperm (i.e., maturedbut not fertilized). The pre-fertilization oocyte has completed thefirst meiotic division, has released the first polar body and lacks anuclear membrane (the nuclear membrane will not reform untilfertilization occurs; after fertilization, the second meiotic divisionoccurs along with the extrusion of the second polar body and theformation of the male and female pronuclei). Pre-fertilization oocytesmay also be referred to as matured oocytes at metaphase 11 of the secondmelosis.

The terms “unfertilized egg” or “unfertilized oocyte” as used herein,refers to any female gamete cell which has not been fertilized and theseterms encompass both pre-maturation and pre-fertilization oocytes.

The term “zygote” as used herein, refers to a fertilized oocyte that hasnot yet undergone the first cleavage step in the development of anembryo (i.e., it is at the single-cell stage).

The term “perivitelline space” refers to the space located between thezona pellucida and the plasma membrane of a mammalian egg or oocyte.

As used herein, the term “trans” is used in reference to the positioningof genes of interest on the different strands of nucleic acid (e.g.,alleles present on the two chromosomes of a chromosomal pair). The term“trans-acting” is used in reference to the controlling effect of aregulatory gene on a gene present on a different chromosome. In contrastto promoters, repressors are not limited in their binding to the DNAmolecule that includes their genetic information. Therefore, repressorsare sometimes referred to as trans-acting control elements.

The term “trans-activation” as used herein refers to the activation ofgene sequences by factors encoded by a regulatory gene which is notnecessarily contiguous with the gene sequences which it binds to andactivates.

As used herein, the term “cis” is used in reference to the presence ofgenes on the same chromosome. The term “cis-acting” is used in referenceto the controlling effect of a regulatory gene on a gene present on thesame chromosome. For example, promoters, which affect the synthesis ofdownstream mRNA are cis-acting control elements.

As used herein, the term “retrovirus” is used in reference to RNAviruses which utilize reverse transcriptase during their replicationcycle (i.e., retroviruses are incapable of replication; rather, theseare useful RNA sequences that are packaged with at least two enzymesthat are required for the insertion of the RNA sequences into the hostcell genome). The retroviral genomic RNA is converted intodouble-stranded DNA by reverse transcriptase. This double-stranded DNAform of the virus integrates into the chromosome of the infected celland is referred to as a “provirus.” In preferred embodiments of thepresent invention, the term “proviral” is used in reference toconstructs that are similar to “retrotransposons.” These are integratedgenes that are bracketed by LTRs in the host cell genome. However, inpreferred embodiments, the proviral constructs cannot replicate. Incontrast, in wild-type viruses, the provirus serves as a template forRNA polymerase 11 and directs the expression of RNA molecules whichencode the structural proteins and enzymes needed to produce new viralparticles. At each end of the provirus are structures called “longterminal repeats” or “LTRs”. The LTR contains numerous regulatorysignals including transcriptional control elements, polyadenylationsignals and sequences needed for replication and integration of theviral genome. The viral LTR is divided into three regions called U3, Rand U5.

The U3 region contains the enhancer and promoter elements. The U5 regioncontains the polyadenylation signals. The R (repeat) region separatesthe U3 and U5 regions and transcribed sequences of the R region appearat both the 5′ and 3′ ends of the viral RNA

As used herein, the term “provirus” is used in reference to a virus thatis integrated into a host cell chromosome (or genome), and istransmitted from one cell generation to the next, without causing lysisor destruction of the host cell. The term is also used in reference to aduplex DNA sequence present in an eukaryotic chromosome, whichcorresponds to the genome of an RNA retrovirus.

As used herein, the term “endogenous virus” is used in reference to aninactive virus which is integrated into the chromosome of its host cell(often in multiple copies), and can thereby exhibit verticaltransmission. Endogenous viruses can spontaneously express themselvesand may result in malignancies.

As used herein, the terms “amphotrope” and “amphotropic” are used inreference to endogenous viruses that readily multiply in cells of thespecies in which they were induced, as well as cells of other species.

As used herein, the term “ecotrope” and “ecotropic” are used inreference to endogenous viruses that multiply readily in cells of thespecies in which they were induced, but cannot multiply in cells ofother species.

As used herein, the term “xenotrope” and “xenotropic” are used inreference to endogenous viruses that cannot infect cells of the speciesin which they were induced, but can infect and multiply in cells ofother species.

The term “Infectious retrovirus” refers to a retroviral particle whichis capable of entering a cell (i.e., the particle contains amembrane-associated protein such as an envelope protein or a viral Gglycoprotein which can bind to the host cell surface and facilitateentry of the viral particle into the cytoplasm of the host cell) andintegrating the retroviral genome (as a double-stranded provirus) intothe genome of the host cell.

As used herein, the term “retroviral vector” is used in reference toretroviruses which have been modified so as to serve as vectors forintroduction of nucleic acid into cells.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another.Retroviral vectors transfer RNA, which is then reverse transcribed intoDNA. The term “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinantmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

The terms “in operable combination,” “in operable order,” and “operablylinked,” as used herein refer to the linkage of nucleic acid sequencesin such a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

As used herein, the term “protein of interest” refers to any protein forwhich expression is desired. For example, the term encompasses anyrecombinant forms of a protein that is desired. The term “gene ofinterest” refers to any gene that is desired. In particularly preferredembodiments, the gene of interest encodes at least a portion of aprotein of interest.

The term “genetic cassette” as used herein refers to a fragment orsegment of nucleic acid containing a particular grouping of geneticelements. The cassette can be removed and inserted into a vector orplasmid as a single unit.

As used herein, the term “long terminal repeat (LTR)” is used inreference to domains of base pairs located at the ends of retroviralDNA's. These LTRs may be several hundred base pairs in length. LTR'soften provide functions fundamental to the expression of most eukaryoticgenes (e.g., promotion, initiation and polyadenylation of transcripts).

Retroviral vectors can be used to transfer genes efficiently into hostcells by exploiting the viral infectious process. Foreign orheterologous genes cloned (i.e., inserted using molecular biologicaltechniques) into the retroviral genome can be delivered efficiently tohost cells which are susceptible to infection by the retrovirus. Throughwell known genetic manipulations, the replicative capacity of theretroviral genome can be destroyed. The resulting replication-defectivevectors can be used to introduce new genetic material to a cell but theyare unable to replicate. A helper virus or packaging cell line can beused to permit vector particle assembly and egress from the cell.

The terms “vector particle” or “retroviral particle” refer to viral-likeparticles that are capable of introducing nucleic acid into a cellthrough a viral-like entry mechanism.

The host range of a retroviral vector (i.e., the range of cells thatthese vectors can infect) can be altered by including an envelopeprotein from another closely related virus.

As used herein, the term “packaging signal” or “packaging sequence”refers to non-coding sequences located within the retroviral genomewhich are required for insertion of the viral RNA into the viral capsidor particle. Several retroviral vectors use the minimal packaging signal(also referred to as the psi sequence) needed for encapsidation of theviral genome. This minimal packaging signal encompasses bases 212 to 563of the Mo-MuLV genome (Mann et al., Cell 33:153 [19831).

As used herein, the term “extended packaging signal” or “extendedpackaging sequence” refers to the use of sequences around the psisequence with further extension into the gag gene. In Mo-MuLV, thisextended packaging sequence corresponds to the region encompassing base1039 to base 1906 (Akagi et al., Gene 106:255 [1991]). The frequentlyused M-MULV vector, pLNL6 (Bender et al., J. Virol., 61:1639 [1987]),contains the entire 5˜-region of the genome including an extendedpackaging signal from bases 206 to 1039 of the Moloney murine sarcomavirus genome (numbering from Supplements and Appendices in RNA TuniorViruses, 2nd Ed. [1985] pp. 986-988). The inclusion of these additionalpackaging sequences increases the efficiency of insertion of vector RNAinto viral particles.

As used herein, the term “packaging cell lines” is used in reference tocell lines that express viral structural proteins (e.g., gag, pol andenv), but do not contain a packaging signal.

When retroviral vector DNA is transfected into the cells, it becomesintegrated into the chromosomal DNA and is transcribed, therebyproducing full-length retroviral vector RNA that has a psi- sequence.Under these conditions, only the vector RNA is packaged into the viralcapsid structures These complete, yet replication-defective, virusparticles can then be used to deliver the retroviral vector to targetcells with relatively high efficiency.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known in the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics. Incontrast, as used herein, the term “transduction” refers to the deliveryof a gene(s) using a retroviral vector by means of infection rather thanby transfection.

The term “membrane-associated protein” refers to a protein (e.g., aviral envelope glycoprotein or the G proteins of viruses in theRhabdoviridae family such as VSV, Piry, Chandipura and Mokola) which areassociated with the membrane surrounding a viral particle; thesemembrane-associated proteins mediate the entry of the viral particleinto the host cell. The membrane associated protein may bind to specificcell surface protein receptors, as is the case for retroviral envelopeproteins or the membrane-associated protein may interact with aphospholipid component of the plasma membrane of the host cell, as isthe case for the G proteins derived from members of the Rhabdoviridaefamily.

The term “heterologous membrane-associated protein” refers to amembrane-associated protein which is derived from a virus which is not amember of the same viral class or family as that from which thenucleocapsid protein of the vector particle is derived. “Viral class orfamily” refers to the taxonomic rank of class or family, as assigned bythe International Committee on Taxonomy of Viruses.

The term “Rhabdoviridae” refers to a family of enveloped RNA virusesthat infect animals, including humans, and plants. The Rhabdoviridaefamily encompasses the genus Vesiculovirus which includes vesicularstomatitis-virus (VSV), Cocal virus, Piry virus, Chandipura virus, andSpring viremia of carp virus (sequences encoding the Spring viremia ofcarp virus are available under GenBank accession number U18101). The Gproteins of viruses in the Vesiculovirus genera are virally-encodedintegral membrane proteins that form externally projecting homotrimericspike glycoproteins complexes that are required for receptor binding andmembrane fusion. The G proteins of viruses in the Vesiculovirus generahave a covalently bound palmitic acid (C₁₆) moiety. The amino acidsequences of the G proteins from the Vesiculoviruses are fairly wellconserved. For example, the Piry virus G protein share about 38%identity and about 55% similarity with the VSV G proteins (severalstrains of VSV are known, e.g., Indiana, New Jersey, Orsay, San Juan,etc., and their G proteins are highly homologous). The Chandipura virusG protein and the VSV G proteins share about 37% identity and 52%similarity. Given the high degree of conservation (amino acid sequence)and the related functional characteristics (e.g., binding of the virusto the host cell and fusion of membranes, including syncytia formation)of the G proteins of the Vesiculoviruses, the G proteins from non-VSVVesiculoviruses may be used in place of the VSV G protein for thepseudotyping of viral particles. The G proteins of the Lyssa viruses(another genera within the Rhabdoviridae family) also share a fairdegree of conservation with the VSV G proteins and function in a similarmanner (e.g., mediate fusion of membranes) and therefore may be used inplace of the VSV G protein for the pseudotyping of viral particles. TheLyssa viruses include the Mokola virus and the Rabies viruses (severalstrains of Rabies virus are known and their G proteins have been clonedand sequenced). The Mokola virus G protein shares stretches of homology(particularly over the extracellular and transmembrane domains) with theVSV G proteins which show about 31% identity and 48% similarity with theVSV G proteins. Preferred G proteins share at least 25% identity,preferably at least 30% identity and most preferably at least 35%identity with the VSV G proteins. The VSV G protein from which NewJersey strain (the sequence of this G protein is provided in GenBankaccession numbers M27165 and M21557) is employed as the reference VSV Gprotein. The term “conditions which permit the maturation of apre-maturation oocyte” refers to conditions of in vitro cell culturewhich permit the maturation of a pre-maturation oocyte to a mature ovum(e.g., a pre-fertilization oocyte). These culture conditions permit andinduce the events which are associated with maturation of thepre-maturation oocyte including stimulation of the first and secondmeiotic divisions. In vitro culture conditions which permit thematuration of pre-maturation oocytes from a variety of mammalian species(e.g., cattle, hamster, pigs and goats) are well know to the art (Seee.g., Parrish et al., Therlogenol., 24:537 [19851; Rosenkrans and First,J. Anim. Sci., 72:434 [1994]; Bavister and Yanagimachi, Biol. Reprod.,16:228 [1977]; Bavister et al., Biol. Reprod., 28:235 [1983]; Leibfriedand Bavister, J. Reprod. Fert., 66:87 [1982]; Keskintepe et al., Zygote2:97 [1994]; Funahashi et al., I Reprod. Fert., 101:159 [19941; andFunahashi et al., Biol. Reprod 50:1072 [1994].

As used herein, the term “remedial gene” refers to a gene whoseexpression is desired in a cell to correct an error in cellularmetabolism, to inactivate a pathogen or to kill a cancerous cell.

As used herein, the term “selectable marker” refers to the use of a genewhich encodes an enzymatic activity that confers resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers may be “dominant”; a dominant selectablemarker encodes an enzymatic activity which can be detected in anyeukaryotic cell line. Examples of dominant selectable markers includethe bacterial aminoglycoside 3′ phosphotransferase gene (also referredto as the neo gene) which confers resistance to the drug G418 inmammalian cells, the bacterial hygromycin G phosphotransferase (hyg)gene which confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpi gene) which confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that there use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene which is used in conjunction withtk cell lines, the CAD gene which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withhprt- cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NewYork (1989) pp.16.9-16.15.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods which depend upon binding between nucleicacids.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the Tm of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueoussolution at I M NaCI (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample which is analyzed for the presence of “target”(defined below). In contrast,“background template” is used in referenceto nucleic acid other than sample template which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, which is capable ofhybridizing to another oligonucleotide of interest. Probes are useful inthe detection, identification and isolation of particular genesequences. It is contemplated that any probe used in the presentinvention will be labelled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochernical assays),fluorescent, radioactive, and luminescent systems. It is furthercontemplated that the oligonucleotide of interest (i.e., to be detected)will be labelled with a reporter molecule. It is also contemplated thatboth the probe and oligonucleotide of interest will be labelled. It isnot intended that the present invention be limited to any particulardetection system or label.

As used herein, the term “target” refers to the region of nucleic acidbounded by the primers used for polymerase chain reaction. Thus, the“target” is sought to be sorted out from other nucleic acid sequences. A“segment” is defined as a region of nucleic acid within the targetsequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, allof which are hereby incorporated by reference, directed to methods forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are the to be“PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of “P-labeled deoxynucleotide triphosphates, such as dCTPor DATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR process itself are, themselves, efficient templates for subsequentPCR amplifications.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of QP replicase, MDV-I RNA is the specific template for thereplicase (Kacian et al., Proc. Nat. Acad. Sci USA 69:3038 [1972]).Other nucleic acid will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (M. Chamberlin et.al.,Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides where there is a mismatch betweenthe oligonucleotide substrate and the template at the ligation junction(Wu and Wallace, Genomics 4:560 [1989]). Finally, thermostablepolymerases, such as Taq and Pfii, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences.

Some amplification techniques take the approach of amplifying and thendetecting target; others detect target and then amplify probe.Regardless of the approach, nucleic acid must be free of inhibitors foramplification to occur at high efficiency.

As used herein, the terms “PCR product” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

As used herein, the term “nested primers” refers to primers that annealto the target sequence in an area that is inside the annealingboundaries used to start PCR (Mullis, et al., Cold Spring HarborSymposia, Vol. 11, pp.263-273 [1986]). Because the nested primers annealto the target inside the annealing boundaries of the starting primers,the predominant PCR-amplified product of the starting primers isnecessarily a longer sequence, than that defined by the annealingboundaries of the nested primers. The PCR-arnplified product of thenested primers is an amplified segment of the target sequence thatcannot, therefore, anneal with the starting primers. Advantages to theuse of nested primers include the large degree of specificity, as wellas the fact that a smaller sample portion may be used and yet obtainspecific and efficient amplification.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleoside triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotidesequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule which is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “Y end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a DNA sequence comprising the codingregion of a gene or in other words the DNA sequence which encodes a geneproduct. The coding region may be present in either a CDNA or genomicDNA form. Suitable control elements such as enhancers/promoters, splicejunctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “transcription unit” refers to the segment ofDNA between the sites of initiation and termination of transcription andthe regulatory elements necessary for the efficient initiation andtermination. For example, a segment of DNA comprising anenhancer/promoter, a coding region and a termination and polyadenylationsequence comprises a transcription unit.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis et al., Science 236:1237 [1987]). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements, i.e., promoters, are also found inprokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview see Voss et al., Trends Biochem. Sci., 11:287 [1986]; andManiatis et al., supra [1987]). For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells (Dijkema et al., EMBO J., 4:761 [1985]). Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor la gene(Uetsuki ef al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene91:217 [1990]; and Mizushima and Nagata, Nue. Acids. Res., 18:5322[1990]) and the long terminal repeats of the Rous sarcoma virus (Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]) and the humancytomegalovirus (Boshart et al., Cell 41:521 [1985]).

As used herein, the term “promoter/enhancer” denotes a segment of DNAwhich contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” orflexogenous” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

The term “factor” refers to a protein or group of proteins necessary forthe transcription or replication of a DNA sequence. For example, SV40 Tantigen is a replication factor which is necessary for the replicationof DNA sequences containing the SV40 origin of replication.Transcription factors are proteins which bind to regulatory elementssuch as promoters and enhancers and facilitate the initiation oftranscription of a gene.

Promoters and enhancers may bind to specific factors which increase therate of activity from the promoter or enhancer. These factors may bepresent in all cell types or may be expressed in a tissue-specificmanner or in virus infected cells. In the absence of such a factor thepromoter may be inactive or may produce a low level of transcriptionalactivity. Such a low level of activity is referred to as a baseline or“basal” rate of activity. Additionally, viral promoter and enhancers maybind to factors encoded by the virus such that the viral promoter orenhancer is “activated” in the presence of the viral factor (in a virusinfected cell or in a cell expressing the viral factor). The level ofactivity in the presence of the factor (i.e., activity “induced” by thefactor) will be higher than the basal rate.

Different promoters may have different levels of basal activity in thesame or different cell types. When two different promoters are comparedin a given cell type in the absence of any inducing factors, if onepromoter expresses at a higher level than the other it is said to have ahigher basal activity.

The activity of a promoter and/or enhancer is measured by detectingdirectly or indirectly the level of transcription from the element(s).Direct detection involves quantitating the level of the RNA transcriptsproduced from that promoter and/or enhancer. Indirect detection involvesquantitation of the level of a protein, often an enzyme, produced fromRNA transcribed from the promoter and/or enhancer. An commonly employedassay for promoter or enhancer activity utilizes the chloramphenicolacetyltransferase (CAT) gene. A promoter and/or enhancer is insertedupstream from the coding region for the CAT gene on a plasmid; theplasmid is introduced into a cell line. The levels of CAT enzyme aremeasured. The level of enzymatic activity is proportional to the amountof CAT RNA transcribed by the cell line. This CAT assay therefore allowsa comparison to be made of the relative strength of different promotersor enhancers in a given cell line. When a promoter is said to express at“high” or “low” levels in a cell line this refers to the level ofactivity relative to another promoter which is used as a reference orstandard of promoter activity.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (See e.g., Sambrook, J.et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, New York [1989], pp. 16.7-16.8). A commonlyused splice donor and acceptor site is the splice junction from the 16SRNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are 10 rapidlydegraded. The poly A signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly A signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly A signal is one which is one which isisolated from one gene and placed 3′ of another gene. A commonly usedheterologous poly A signal is the SV40 poly A signal. The SV40 poly Asignal is contained on a 237 bp Bam HUM I restriction fragment anddirects both termination and polyadenylation (Sambrook, J., supra, at16.6-16.7).

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequenceswhich allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors whichcontain either the SV40 or polyoma virus origin of replication replicateto high copy number (up to 104 copies/cell) in cells that express theappropriate viral antigen. Vectors which contain the replicons frombovine papillornavirus or Epstein-Barr virus replicateextrachromosomally at low copy number (−100 copies/cell).

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

As used herein, the term “stably maintained” refers to characteristicsof recombinant (i.e., transgenic) animals that maintain at least one oftheir recombinant elements (i.e., the element that is desired) throughmultiple generations. For example, it is intended that the termencompass the characteristics of transgenic animals that are capable ofpassing the transgene to their offspring, such that the offspring arecapable of maintaining the expression and/or transcription of thetransgene. It is not intended that the term be limited to any particularorganism or any specific recombinant element.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells which have taken up foreign DNAbut have failed to integrate this DNA.

As used herein, the term “gene of interest” refers to the gene insertedinto the polylinker of an expression vector. When the gene of interestencodes a gene which provides a therapeutic function, the gene ofinterest may be alternatively called a remedial gene.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the term “adoptive transfer” is used in reference to thetransfer of one function to another cell or organism. For example, in“adoptive immunity,” transfer of an immune function is made from oneorganism to another through the transfer of immunologically competentcells.

DESCRIPTION OF THE INVENTION

The present invention provides improved methods for the production oftransgenic animals. The methods of the present invention provide, forthe first time, the production of transgenic animals by the introductionof exogenous DNA into pre-maturation oocytes and mature, unfertilizedoocytes (i.e., pre-fertilization oocytes) using retroviral vectors whichtransduce dividing cells (e.g., vectors derived from murine leukemiavirus [MLV]). In addition, the present invention provides methods andcompositions for cytomegalovirus promoter-driven, as well as mousemammary tumor LTR expression of various recombinant proteins.

For example, the human cytornegalovirus (CMV) promoter has beendeveloped for use in retroviral vectors for driving the expression ofvarious recombinant proteins, and cell lines have been infected withthese vectors, with resultant recombinant protein expression. Inaddition, the mouse mammary tumor virus (MMTV) LTR has been previouslyshown to control expression of a recombinant protein in transgenic mice(Yom el al., Animal Biotech., 4:89-107 [1993]). In these mouse lines,expression was predominately observed in the mammary gland and milk, butlow expression was also observed in the salivary gland, spleen, lung andkidney. The transgenic mice used in this experiment were produced usingtypical microinjection techniques. In contrast, the present inventionprovides methods and compositions for the use of MMTV LTR-drivenexpression which avoids the need for microinjection techniques. Forexample, the MMTV LTR has been developed for use in retroviral vectorsfor driving the expression of various recombinant proteins, and celllines have been infected with these vectors, with resultant recombinantprotein expression.

The following Description of the Invention is divided into the followingsections: I. Retroviruses and Retroviral Vectors; II. Integration ofRetroviral DNA; III. Introduction of Retroviral Vectors Into GametesBefore the Last Melotic Division; IV. Detection of the RetrovirusFollowing Injection Into Oocytes or Embryos; and V. Expression ofForeign Proteins in Transgenic Animals.

I. Retroviruses and Retroviral Vectors

Retroviruses (family Retroviridae) are divided into three groups: thespurnaviruses (e.g., human foamy virus); the lentiviruses (e.g., humanimmunodeficiency virus and sheep visna virus) and the oncoviruses (e.g.,MLV, Rous sarcoma virus).

Retroviruses are enveloped (i.e., surrounded by a host cell-derivedlipid bilayer membrane) single-stranded RNA viruses which infect animalcells. When a retrovirus infects a cell, its RNA genome is convertedinto a double-stranded linear DNA form (i.e., it is reversetranscribed). The DNA form of the virus is then integrated into the hostcell genorne as a provirus. The provirus serves as a template for theproduction of additional viral genomes and viral mRNAs. Mature viralparticles containing two copies of genomic RNA bud from the surface ofthe infected cell. The viral particle comprises the genomic RNA, reversetranscriptase and other pol gene products inside the viral capsid (whichcontains the viral gag gene products) which is surrounded by a lipidbilayer membrane derived from the host cell containing the viralenvelope glycoproteins (also referred to as membrane-associatedproteins).

The organization of the genomes of numerous retroviruses is well knownin the art and this has allowed the adaptation of the retroviral genometo produce retroviral vectors. The production of a recombinantretroviral vector carrying a gene of interest is typically achieved intwo stages. First, the gene of interest is inserted into a retroviralvector which contains the sequences necessary for the efficientexpression of the gene of interest (including promoter and/or enhancerelements which may be provided by the viral long terminal repeats [LTRs]or by an internal promoter/enhancer and relevant splicing signals),sequences required for the efficient packaging of the viral RNA intoinfectious virions (e.g., the packaging signal [Psi], the tRNA primerbinding site [−PBS], the 3′ regulatory sequences required for reversetranscription [+PBS] and the viral LTRs). The LTRs contain sequencesrequired for the association of viral genomic RNA, reverse transcriptaseand integrase functions, and sequences involved in directing theexpression of the genomic RNA to be packaged in viral particles. Forsafety reasons, many recombinant retroviral vectors lack functionalcopies of the genes which are essential for viral replication (theseessential genes are either deleted or disabled); the resulting virus issaid to be replication defective.

Second, following the construction of the recombinant vector, the vectorDNA is introduced into a packaging cell line. Packaging cell linesprovide viral proteins required in trans for the packaging of the viralgenomic RNA into viral particles having the desired host range (i.e.,the viral-encoded gag, pol and env proteins). The host range iscontrolled, in part, by the type of envelope gene product expressed onthe surface of the viral particle. Packaging cell lines may expressecotrophic, amphotropic or xenotropic envelope gene products.Alternatively, the packaging cell line may lack sequences encoding aviral envelope (env) protein. In this case the packaging cell line willpackage the viral genome into particles which lack a membrane-associatedprotein (e.g., an env protein). In order to produce viral particlescontaining a membrane associated protein which will permit entry of thevirus into a cell, the packaging cell line containing the retroviralsequences is transfected with sequences encoding a membrane-associatedprotein (e.g., the G protein of vesicular stomatitis virus [VSV]). Thetransfected packaging cell will then produce viral particles whichcontain the membrane-associated protein expressed by the transfectedpackaging cell line; these viral particles which contain viral genomicRNA derived from one virus encapsidated by the envelope proteins ofanother virus are said to be pseudotyped virus particles. oftransferring genes into cells as compared to other techniques such ascalcium phosphate-DNA co-precipitation or DEAE-dextran-mediatedtransfection, electroporation or microinjection of nucleic acids. It isbelieved that the efficiency of viral transfer is due in part to thefact that the transfer of nucleic acid is a receptor-mediated process(i.e., the virus binds to a specific receptor protein on the surface ofthe cell to be infected). In addition, the virally transferred nucleicacid once inside a cell integrates in controlled manner in contrast tothe integration of nucleic acids which are not virally transferred;nucleic acids transferred by other means such as calcium phosphate-DNAco-precipitation are subject to rearrangement and degradation.

The most commonly used recombinant retroviral vectors are derived fromthe amphotropic Moloney murine leukemia virus (MoMLV) (Miller andBaltimore, Mol. Cell. Biol., 6:2895 [1986]). The MOMLV system hasseveral advantages: 1) this specific retrovirus can infect manydifferent cell types, 2) established packaging cell lines are availablefor the production of recombinant MOMLV viral particles and 3) thetransferred genes are permanently integrated into the target cellchromosome. The established MOMLV vector systems comprise a DNA vectorcontaining a small portion of the retroviral sequence (the viral longterminal repeat or “LTR” and the packaging or “psi” signal) and apackaging cell line. The gene to be transferred is inserted into the DNAvector. The viral sequences present on the DNA vector provide thesignals necessary for the insertion or packaging of the vector RNA intothe viral particle and for the expression of the inserted gene. Thepackaging cell line provides the viral proteins required for particleassembly (Markowitz et al., J. Virol., 62:1120 [1988]).

Despite these advantages, existing retroviral vectors based upon MOMLVare limited by several intrinsic problems: 1) they do not infectnon-dividing cells (Miller et al., Mol. Cell. Biol., 10:4239 [1992]), 2)they produce low titers of the recombinant virus (Miller and Rosman,BioTechn., 7: 980 [1989]; and Miller, Nature 357: 455 [1992]) and 3)they infect certain cell types (e.g., human lymphocytes) with lowefficiency (Adams et al., Proc. Natl. Acad. Sci. USA 89:8981 [1992]).The low titers associated with MoMLV-based vectors has been attributed,at least in part, to the instability of the virus-encoded envelopeprotein. Concentration of retrovirus stocks by physical means (e.g.,ultracentrifugation and ultrafiltration) leads to a severe loss ofinfectious virus.

The low titer and inefficient infection of certain cell types byMoMLV-based vectors has been overcome by the use of pseudotypedretroviral vectors which contain the G protein of VSV as the membraneassociated protein. Unlike retroviral envelope proteins which bind to aspecific cell surface protein receptor to gain entry into a cell, theVSV G protein interacts with a phospholipid component of the plasmamembrane (Mastromarino et al., J. Gen. Virol., 68:2359 [1977]). Becauseentry of VSV into a cell is not dependent upon the presence of specificprotein receptors, VSV has an extremely broad host range. Pseudotypedretroviral vectors bearing the VSV G protein have an altered host rangecharacteristic of VSV (i.e., they can infect almost all species ofvertebrate, invertebrate and insect cells). Importantly, VSVG-pseudotyped retroviral vectors can be concentrated 2000-fold or moreby ultracentrifugation without significant loss of infectivity (Burns etal., Proc. Natl. Acad. Sci. USA 90:8033 [1993]).

The VSV G protein has also been used to pseudotype retroviral vectorsbased upon the human immunodeficiency virus (HIV) (Naldini et al.,Science 272:263 [1996]). Thus, the VSV G protein may be used to generatea variety of pseudotyped retroviral vectors and is not limited tovectors based on MoMLV.

The present invention is not limited to the use of the VSV G proteinwhen a viral G protein is employed as the heterologousmembrane-associated protein within a viral particle. The G proteins ofviruses in the Vesiculovirus genera other than VSV, such as the Piry andChandipura viruses, that are highly homologous to the VSV G protein and,like the VSV G protein, contain covalently linked palmitic acid (Brun etal., Intervirol., 38:274 [1995]; and Masters et al., Virol., 171:285[1990]). Thus, the G protein of the Piry and Chandipura viruses can beused in place of the VSV G protein for the pseudotyping of viralparticles. In addition, the VSV G proteins of viruses within the Lyssavirus genera such as Rabies and Mokola viruses show a high degree ofconservation (amino acid sequence as well as functional conservation)with the VSV G proteins. For example, the Mokola virus G protein hasbeen shown to function in a manner similar to the VSV G protein (i.e.,to mediate membrane fusion) and therefore may be used in place of theVSV G protein for the pseudotyping of viral particles (Mebatsion et al.,J. Virol., 69:1444 [1995]). The nucleotide sequence encoding the Piry Gprotein is provided in SEQ ID NO:5 and the amino acid sequence of thePiry G protein is provided in SEQ ID NO:6. The nucleotide sequenceencoding the Chandipura G protein is provided in SEQ ID NO:7 and theamino acid sequence of the Chandipura G protein is provided in SEQ IDNOX The nucleotide sequence encoding the Mokola G protein is provided inSEQ ID NO:9 and the amino acid sequence of the Mokola G protein isprovided in SEQ rD NO:10. Viral particles may be pseudotyped usingeither the Piry, Chandipura or Mokola G protein as described in Example2 with the exception that a plasmid containing sequences encoding eitherthe Piry, Chandipura or Mokola G protein under the transcriptionalcontrol of a suitable promoter element (e.g., the CMV intermediate earlypromoter; numerous expression vectors containing the CMV IE promoter areavailable, such as the pcDNA3.1 vectors [Invitrogen]) is used in placeof pHCMV-G. Sequences encoding other G proteins derived from othermembers of the Rhabdoviridae family may be used; sequences encodingnumerous rhabdoviral G proteins are available from the GenBank database.

II. Integration of Retroviral DNA

The majority of retroviruses can transfer or integrate a double-strandedlinear form of the virus (the provirus) into the genome of the recipientcell only if the recipient cell is cycling (i.e., dividing) at the timeof infection. Retroviruses which have been shown to infect dividingcells exclusively, or more efficiently, include MLV, spleen necrosisvirus, Rous sarcoma virus and human immunodeficiency virus (HIV; whileHIV infects dividing cells more efficiently, HIV can infect non-dividingcells).

It has been shown that the integration of MLV virus DNA depends upon thehost cell's progression through mitosis and it has been postulated thatthe dependence upon mitosis reflects a requirement for the breakdown ofthe nuclear envelope in order for the viral integration complex to gainentry into the nucleus (Roe et al., EMBO J., 12:2099 [1993]). However,as integration does not occur in cells arrested in metaphase, thebreakdown of the nuclear envelope alone may not be sufficient to permitviral integration; there may be additional requirements such as thestate of condensation of the genomic DNA (Roe et al.,supra).

III. Introduction of Retroviral Vectors Into Gametes Before the LastMeiotic Division

The nuclear envelope of a cell breaks down during melosis as well asduring mitosis. Meiosis occurs only during the final stages ofgametogenesis. The methods of the present invention exploit thebreakdown of the nuclear envelope during meiosis to permit theintegration of recombinant retroviral DNA and permit for the first timethe use of unfertilized oocytes (i.e., pre-fertilization andpre-maturation oocytes) as the recipient cell for retroviral genetransfer for the production of transgenic animals. Because infection ofunfertilized oocytes permits the integration of the recombinant provirusprior to the division of the one cell embryo, all cells in the embryowill contain the proviral sequences.

Oocytes which have not undergone the final stages of gametogenesis areinfected with the retroviral vector. The injected oocytes are thenpermitted to complete maturation with the accompanying meloticdivisions. The breakdown of the nuclear envelope during meiosis permitsthe integration of the proviral form of the retrovirus vector into thegenome of the oocyte. When pre-maturation oocytes are used, the injectedoocytes are then cultured in vitro under conditions which permitmaturation of the oocyte prior to fertilization in vitro. Conditions forthe maturation of oocytes from a number of mammalian species (e.g.,bovine, ovine, porcine, murine, caprine) are well known to the art. Ingeneral, the base medium used herein for the in vitro maturation ofbovine oocytes, TC-M199 medium, may be used for the in vitro maturationof other mammalian oocytes. TC-M199 medium is supplemented with hormones(e.g., luteinizing hormone and estradiol) from. the appropriatemammalian species. The amount of time a pre-maturation oocyte must beexposed to maturation medium to permit maturation varies betweenmammalian species as is known to the art. For example, an exposure ofabout 24 hours is sufficient to permit maturation of bovine oocyteswhile porcine oocytes require about 44-48 hours.

Oocytes may be matured in vivo and employed in place of oocytes maturedin vitro in the practice of the present invention. For example, whenporcine oocytes are to be employed in the methods of the presentinvention, matured pre-fertilization oocytes may be harvested directlyfrom pigs that are induced to superovulate as is known to the art.Briefly, on day 15 or 16 of estrus the female pig(s) is injected withabout 1000 units of pregnant mare's serum (PMS; available from Sigma andCalbiochem). Approximately 48 hours later, the pig(s) is injected withabout 1000 units of human chorionic gonadotropin) (hCG; Sigma) and 24-48hours later matured oocytes are collected from oviduct. These in vivomatured pre-fertilization oocytes are then injected with the desiredretroviral preparation as described herein. Methods for thesuperovulation and collection of in vivo matured (i.e., oocytes at themetaphase 2 stage) oocytes are known for a variety of mammals (e.g., forsuperovulation of mice, see Hogan et aL, supra at pp. 130-133 [1994];for superovulation of pigs and in vitro fertilization of pig oocytes seeCheng, Doctoral Dissertation, Cambridge University, Cambridge, UnitedKingdom [1995]).

Retroviral vectors capable of infecting the desired species of non-humananimal which can be grown and concentrated to very high titers (e.g., ≧:1×10⁸ cfu/ml) are preferentially employed. The use of high titer virusstocks allows the introduction of a defined number of viral particlesinto the perivitelline space of each injected oocyte. The perivitellinespace of most mammalian oocytes can accommodate about 10 picoliters ofinjected fluid (those in the art know that the volume that can beinjected into the perivitelline space of a mammalian oocyte or zygotevaries somewhat between species as the volume of an oocyte is smallerthan that of a zygote and thus, oocytes can accommodate somewhat lessthan can zygotes).

The vector used may contain one or more genes encoding a protein ofinterest; alternatively, the vector may contain sequences which produceanti-sense RNA sequences or ribozymes. The infectious virus ismicroinjected into the perivitelline space of oocytes (includingpre-maturation oocytes) or one cell stage zygotes. Microinjection intothe perivitelline space is much less invasive than the microinjection ofnucleic acid into the pronucleus of an embryo. Pronuclear injectionrequires the mechanical puncture of the plasma membrane of the embryoand results in lower embryo viability. In addition, a higher level ofoperator skill is required to perform pronuclear injection as comparedto perivitelline injection. Visualization of the pronucleus is notrequired when the virus is injected into the perivitelline space (incontrast to injection into the pronucleus); therefore injection into theperivitelline space obviates the difficulties associated withvisualization of pronuclei in species such as cattle, sheep and pigs.

The virus stock may be titered and diluted prior to microinjection intothe perivitelline space so that the number of proviruses integrated inthe resulting transgenic animal is controlled. The use of a viral stock(or dilution thereof) having a titer of 1×10⁸ cfu/ml allows the deliveryof a single viral particle per oocyte. The use of pre-maturation oocytesor mature fertilized oocytes as the recipient of the virus minimizes theproduction of animals which are mosaic for the provirus as the virusintegrates into the genome of the oocyte prior to the occurrence of cellcleavage.

In order to deliver, on average, a single infectious particle peroocyte, the micropipets used for the injection are calibrated asfollows. Small volumes (e.g., about 5-10 pl) of the undiluted high titerviral stock (e.g., a titer of about 1×10⁸ cfU/ml) are delivered to thewells of a microtiter plate by pulsing the micromanipulator. The titerof virus delivered per a given number of pulses is determined bydiluting the viral stock in each well and determining the titer using asuitable cell line (e.g., the 208F cell line) as described in Ex. 2. Thenumber of pulses which deliver, on average, a volume of virus stockcontaining one infectious viral particle (i.e., gives a MOI of 1 whentitered on 208F cells) are used for injection of the viral stock intothe oocytes.

Prior to microinjection of the titered and diluted (if required) virusstock, the cumulus cell layer is opened to provide access to theperivitelline space. The cumulus cell layer need not be completelyremoved from the oocyte and indeed for certain species of animals (e.g.,cows, sheep, pigs, mice) a portion of the cumulus cell layer must remainin contact with tile oocyte to permit proper development andfertilization post-injection. Injection of viral particles into theperivitelline space allows the vector RNA (i.e., the viral genome) toenter the cell through the plasma membrane thereby allowing properreverse transcription of the viral RNA.

IV. Detection of the Retrovirus Following Injection Into Oocytes orEmbryos

The presence of the retroviral genome in cells (e.g., oocytes orembryos) infected with pseudotyped retrovirus may be detected using avariety of means. The expression of the gene product(s) encoded by theretrovirus may be etected by detection of mRNA corresponding to thevector-encoded gene products using techniques well known to the art(e.g., Northern blot, dot blot, in situ hybridization and RT-PCRanalysis). Direct detection of the vector-encoded gene product(s) isemployed when the gene product is a protein which either has anenzymatic activity (e.g., P-galactosidase) or when an antibody capableof reacting with the vector encoded protein is available.

Alternatively, the presence of the integrated viral genome may bedetected using Southern blot or PCR analysis. For example, the presenceof the LZRNL or LSRNL genomes may be detected following infection ofoocytes or embryos using PCR as follows. Genomic DNA is extracted fromthe infected oocytes or embryos (the DNA may be extracted from the wholeembryo or alternatively various tissues of the embryo may be examined)using techniques well known to the art. The LZRNL and LSRNL virusescontain the neo gene and the following primer pair can be used toamplify a 349-bp segment of the neo gene:upstream primer:5′-GCATTGCATCAGCCATGATG-3′ (SEQ ID NO: I) and downstream primer:5′-GATGGATTGCACGCAGGTTC-3′ (SEQ ID NO:2). The PCR is carried out usingwell known techniques (e.g., using a GeneAmp kit according to themanufacturer's instructions [Perkin-Elmer]). The DNA present in thereaction is denatured by incubation at 94° C. for 3 min followed by 40cycles of 94° C. for 1 min, 60° C. for 40 sec and 72° C. for 40 secfollowed by a final extension at 72° C. for 5 min. The PCR products maybe analyzed by electrophoresis of 10 to 20% of the total reaction on a2% agarose gel; the 349-bp product may be visualized by staining of thegel with ethidium bromide and exposure of the stained gel to UV light.If the expected PCR product cannot be detected visually, the DNA can betransferred to a solid support (e.g., a nylon membrane) and hybridizedwith a ³²P-labeled neo probe.

Southern blot analysis of genomic DNA extracted from infected oocytesand/or the resulting embryos, offspring and tissues derived therefrom isemployed when information concerning the integration of the viral DNAinto the host genome is desired. To examine the number of integrationsites present in the host genome, the extracted genomic DNA is typicallydigested with a restriction enzyme which cuts at least once within thevector sequences. If the enzyme chosen cuts twice within the vectorsequences, a band of known (i.e., predictable) size is generated inaddition to two fragments of novel length which can be detected usingappropriate probes.

V. Detection of Foreign Protein Expression in Transgenic Animals

The present invention also provides transgenic animals that are capableof expressing foreign proteins in their milk, urine and blood. Asindicated in Examples 8-10, the transgene is stable, as it is shown tobe passed from a transgenic bull to his offspring (See, Example 8). Inaddition, as shown in Examples 9 and 10, transgenic animals producedaccording to the present invention express foreign proteins in theirbody fluids (e.g., milk, blood, and urine). Thus, these data furtherdemonstrate the utility of using the MOMLV LTR as a promoter for drivingthe constitutive production of foreign proteins in transgenic cattle. Itis also contemplated that such a promoter could be used to controlexpression of proteins that would prevent disease and/or infection inthe transgenic animals and their offspring, or be of use in theproduction of a consistent level of protein expression in a number ofdifferent tissues and body fluids.

For example, it is contemplated that the MOMLV LTR of the presentinvention will find use in driving expression of antibody to pathogenicorganisms, thereby preventing infection and/or disease in transgenicanimals created using the methods of the present invention. For example,it is contemplated that antibodies directed against organisms such as E.coli, Salmonella ssp., Streptococcus ssp., Staphylococcus spp.,Mycobacterium spp., produced by transgenic animals will find usepreventing mastitis, scours, and other diseases that are common problemsin young animals. It is also contemplated that proteins expressed bytransgenic animals produced according to the present invention will finduse as bacteriostatic, bactericidal, fungistatic, fungicidal, viricidal,and/or anti-parasitic compositions. Thus, it is contemplated thattransgenic animals produced according to the present invention will beresistant to various pathogenic organisms. Furthermore, the milkproduced by female transgenic animals would contain substantial antibodylevels. It is contemplated that these antibodies will find use in theprotection of other animals (e.g., through passive immunizationmethods).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM(nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg(picograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); AMP (adenosine 5′-monophosphate); BSA (bovineserum albumin); cDNA (copy or complimentary DNA); CS (calf serum); DNA(deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (doublestranded DNA); DNTP (deoxyribonucleotide triphosphate); LH (luteinizinghormone); NIH (National Institutes of Health, Bethesda, Md.); RNA(ribonucleic acid); PBS (phosphate buffered saline); g (gravity); OD(optical density);HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS(HEPES buffered saline); PBS (phosphate buffered saline); SDS (sodiumdodecyl sulfate); Tris-HCI(tris[Hydroxymethyl]aminomethane-hydrochloride); Klenow (DNA polymeraseI large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethyleneglycol-bis(β-aminoethyl ether) N,N,N′, N′-tetraacetic acid); EDTA(ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); ORI (plasmid origin of replication); lacI(lac repressor); X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside);ATCC (American Type Culture Collection, Rockville, Md.); GIBCO/BRL(GIBCO/BRL, Grand Island, N.Y.); Perkin-Elmer (Perkin-Elmer, Norwalk,Conn.); Abbott (Abbott Laboratories, Diagnostics Division, Abbott Park,Ill. 60064); and Sigma (Sigma Chemical Company, St. Louis, Mo.).

EXAMPLE 1

Generation of Cell Lines Stably Expressing the MoMLV Gag and PolProteins

The expression of the fusogenic VSV G protein on the surface of cellsresults in syncytium formation and cell death. Therefore, in order toproduce retroviral particles containing the VSV G protein as themembrane-associated protein a three step approach was taken. First,stable cell lines expressing the Gag and Pol proteins from MoMLV at highlevels were generated (e.g., 293GP cells; Example 1). These stable celllines were then infected using the desired retroviral vector which isderived from an amphotrophic packaging cell (e.g., PA317 cellstransfected with the desired retroviral vector; Example 2a). Theinfected stable cell line which expresses the Gag and Pol proteinsproduces noninfectious viral particles lacking a membrane-associatedprotein (e.g., a envelope protein). Third, these infected cell lines arethen transiently transfected with a plasmid capable of directing thehigh level expression of the VSV G protein (Example 2b). The transientlytransfected cells produce VSV G-pseudotyped retroviral vectors which canbe collected from the cells over a period of 3 to 4 days before theproducing cells die as a result of syncytium formation.

The first step in the production of VSV G-pseudotyped retroviralvectors, the generation of stable cell lines expressing the MoMLV Gagand Pol proteins is described below.

The human adenovirus 5-transformed embryonal kidney cell line 293 (ATCCCRL 1573) was cotransfected with the pCMVgag-pol and pFR400 plasmidsusing a ratio of 10:1 (pCMVgag-pol and pFR400). pCMV gag-pol containsthe MoMLV gag and pol genes under the control of the CMV promoter (pCMVgag-pol is available from the ATCQ. pFR400 encodes a mutantdihydrofolate reductase which has a reduced affinity for methotrexate(Simonsen et al., Proc. Natl. Acad. Scl. 80:2495 [1983]).

The plasmid DNA was introduced into the 293 cells using calciumphosphate co-precipitation (Graham and Van der Eb, Virol., 52:456[1973]). Approximately 5×10⁵ 293 cells were plated into a 100 mm tissueculture plate the day before the DNA co-precipitate was added. A totalof 20 μg of plasmid DNA (18 μg pCMV gag-pol and 2 μg pFR400) was addedas a calcium-DNA co-precipitate to each 100 min plate. Stabletransformants were selected by growth in DMEM-high glucose mediumcontaining 10% FCS, 0.5 μM methotrexate and 5 μM dipyridimole (i.e.,selective medium). Colonies which grew in the selective medium werescreened for extracellular reverse transcriptase activity (Goff et al.,J. Virol., 38:239 [1981]) and intracellular p30^(gag) expression.p30^(gag) expression was determined by Western blotting using agoat-anti p30 antibody (NCI antiserum 77SO00087). A clone whichexhibited stable expression of the retroviral genes in the absence ofcontinued methotrexate selection was selected. This clone was named293GP (293 gag-pol). The 293GP cell line, a derivative of the humanAd-5-transformed embryonal kidney cell line 293, was grown in DMEM-highglucose medium containing 10% FCS. The 293GP cell line is commerciallyavailable from Viagen, Inc., San Diego, Calif.

EXAMPLE 2

Preparation of Pseudotyped Retroviral Vectors Bearing the G Glycoproteinof VSV

In order to produce VSV G protein pseudotyped retrovirus the followingsteps were taken. First, the 293GP cell line was infected with virusderived from the amphotrophic packaging cell line PA317. The infectedcells packaged the retroviral RNA into viral particles which lack amembrane-associated protein (because the 293GP cell line lacks an envgene or other gene encoding a membrane-associated protein). The infected293GP cells were then transiently transfected with a plasmid encodingthe VSV G protein to produce pseudotyped viral particles bearing the VSVG protein.

a) Cell Lines and Plasmids

The amphotropic packaging cell line, PA317 (ATCC CRL 9078) was grown inDMEM-high glucose medium containing 10% FCS. The 293GP cell line wasgrown in DMEM-high glucose medium containing 10% FCS. The titer of thepseudo-typed virus may be determined using either 208F cells (Quade,Virol., 98:461 [1979]), or NIH/3T3 ) cells (ATCC CRL 1658); 208F andNIH/3T3 cells are grown in DMEM-high glucose medium containing 10% CS.

The plasmid pLZRNL (Xu et al., Virol., 171:331 [1989]) contains the geneencoding E. coli β-galactosidase (LacZ) under the transcriptionalcontrol of the LTR of the Moloney murine sarcoma virus (MSV) followed bythe gene encoding neomycin phosphotransferase (Neo) under thetranscriptional control of the Rous sarcoma virus (RSV) promoter. Theplasmid pLSRNL contains the gene encoding the hepatitis B surfaceantigen gene (HBsAg) under the transcriptional control of the MSV LTRfollowed by the Neo gene under the control of the RSV promoter (U.S.Pat. No. 5,512,421, the disclosure of which is herein incorporated byreference). The plasmid pHCMV-G contains the VSV G gene under thetranscriptional control of the human cytomegalovirus intermediate-earlypromoter (Yee et al. Meth. Cell Biol., 43:99 [1994]).

b) Production and Titering of Pseudotyped LZRNL Virus

pLZRNL DNA was transfected into tne amphotropic packaging line PA317 toproduced LZRNL virus. The resulting LZRNL virus was then used to infect293GP cells to produce pseudotyped LZRNL virus bearing the VSV G protein(following transient transfection of the infected 293GP cells with aplasmid encoding the VSV G protein). The procedure for producingpseudotyped LZRNL virus was carried out as described (Yee et al. Meth.Cell Biol., 43:99 [1994]).

Briefly, on day 1, approximately 5×10⁵ PA317 cells were placed in a 100mm tissue culture plate. On the following day (day 2), the PA317 cellswere transfected with 20 [Ig of pLZRNL plasmid DNA (plasmid DNA waspurified using CsCl gradients) using the standard calcium phosphateco-precipitation procedure (Graham and Van der Eb, Virol., 52:456[1973]). A range of 10 to 40 ug of plasmid DNA may be used. Because293GP cells may take more than 24 hours to attach firmly to tissueculture plates, the 293GP cells may be placed in 100 mm plates 48 hoursprior to transfection. The transfected PA317 cells provide amphotropicLZRNL virus.

On day 3, approximately 1×10⁵ 293GP cells were placed in a 100 nimtissue culture plate 24 hours prior to the harvest of the amphotropicvirus from the transfected PA317 cells. On day 4, culture medium washarvested from the transfected PA317 cells 48 hours after theapplication of the PLZRNL DNA. The culture medium was filtered through a0.45 μm filter and polybrene was added to a final concentration of 8μg/ml. A stock solution of polybrene was prepared by dissolving 0.4 gmhexadimethrine bromide (polybrene; Sigma) in 100 ml sterile water; thestock solution was stored at 40° C. The culture medium containing LZRNLvirus (containing polybrene) was used to infect the 293GP cells asfollows. The culture medium was removed from the 293GP cells and wasreplaced with the LZNRL virus containing culture medium. The viruscontaining medium was allowed to remain on the 293GP cells for 16 hours.Following the 16 hour infection period (on day 5), the medium wasremoved from the 293GP cells and was replaced with fresh mediumcontaining 400 μg/ml G418 (GIBCO/BRL). The medium was changed every 3days until G418-resistant colonies appeared two weeks later. Care wastaken not to disturb the G418-resistant colonies when the medium waschanged as 293GP cells attach rather loosely to tissue culture plates.

The G418-resistant 293 colonies were picked using an automatic pipettorand transferred directly into 24-well plates (i.e., the colonies werenot removed from the plates using trypsin). The G418-resistant 293colonies (as termed “293GP/LZRNL” cells) were screened for theexpression of the LacZ gene in order to identify clones which producehigh titers of pseudotyped LZRNL virus. Clones in 24-well plates weretransferred to 100 mm tissue culture plates and allowed to grow toconfluency. Protein extracts are prepared from the confluent plates bywashing the cells once with 10 ml PBS (137 mM NaCl, 2.6 mM KCI, 8.1 mMNa2HP04, 1.5 mM KH2PO4). Two ml of 250 mM Tris-HCI, pH 7.8 was added andthe cells were scrapped off the plate using a rubber policeman. Thecells were then collected by centrifugation at room temperature andresuspended in 100 μl 250 mM Tris-HCI, pH 7.8. The cells were subjectedto four rapid freeze/thaw cycles followed by centrifugation at roomtemperature to remove cell debris. The β-galactosidase activity presentin the resulting protein extracts was determined as follows. Fivemicroliters of protein extract was mixed with 500 μl β-gal buffer (50 mMTris-HCI, pH 7.5, 100 mM NaCl, 10 mM MgCl₂) containing 0.75 ONPG(Sigma). The mixtures were incubated at 37° C. until a yellow colorappeared. The reactions were stopped by the addition of 500 μl 10 mMEDTA and the optical density of the reactions was determined at 420 nm.

The 293GP/LZRNL clone which generated the highest amount ofP-galactosidase activity was then expanded and used subsequently for theproduction of pseudotyped LZNRL virus as follows. Approximately 1×10⁶293GP/LZRNL cells were placed into a 100 mm tissue culture plate.Twenty-four hours later, the cells were transfected with 20 μg ofpHCMV-G plasmid DNA using calcium phosphate co-precipitation. Six toeight hours after the calcium-DNA precipitate was applied to the cells,the DNA solution was replaced with fresh culture medium (lacking G418).Longer transfection times (overnight) have been found to result in thedetachment of the majority of the 293GP/LZRNL cells from the plate andare therefore avoided. The transfected 293GP/LZRNL cells producepseudotyped LZRNL virus.

The pseudotyped LZRNL virus generated from the transfected 293GP/LZRNLcells can be collected at least once a day between 24 and 96 hr aftertransfection. The highest virus titer was generated approximately 48 to72 hr after initial pHCMV-G transfection. While syncytium formationbecame visible about 48 hr after transfection the majority of thetransfected cells, the cells continued to generate pseudotyped virus forat least an additional 48 hr as long as the cells remained attached tothe tissue culture plate. The collected culture medium containing theVSV G-pseudotyped LZRNL virus was pooled, filtered through a 0.45 μmfilter and stored at −70° C.

The titer of the VSV G-pseudotyped LZRNL virus was then determined asfollows. 5×10⁵ rat 208F fibroblasts or NIH 3T3 cells were plated in a100 min culture plate. Twenty-fours hours after plating, the cells wereinfected with serial dilutions of the LZRNL virus containing culturemedium in the presence of 8 μg/ml polybrene. Sixteen hours afterinfection with virus, the medium was replaced with fresh mediumcontaining 400 μg/ml G418 and selection was continued for 14 days untilG418-resistant colonies became visible. Viral titers were typicallyabout 0.5 to 5.0×10⁶ colony forming units (cfu)/ml. The titer of thevirus stock could be concentrated to a titer of greater than 10⁹ cfu/mlas described below.

EXAMPLE 3

Concentration of Pseudotyped Retroviral Vectors

The VSV G-pseudotyped LZRNL virus was concentrated to a high titer bytwo cycles of ultracentrifugation. The frozen culture medium collectedas described in Example 2 which contained pseudotyped LZRNL virus wasthawed in a 37° C. water bath and was then transferred to ultraclearcentrifuge tubes (14×89 min; Beckman, Palo Alto, Calif.) which had beenpreviously sterilized by exposing the tubes to UV light in a laminarflow hood overnight. The virus was sedimented in a SW41 rotor (Beckman)at 50,000×g (25,000 rpm) at 4° C. for 90 min. The culture medium wasthen removed from the tubes in a laminar flow hood and the tubes werewell drained. The virus pellet was resuspended to 0.5 to 1% of theoriginal volume of culture medium in either TNE (50 mM Tris-HCI, pH 7.8;130 mM NaCl; I mM EDTA) or 0.1X Hank's balanced salt solution (1X Hank'sbalanced salt solution contains 1.3 mM CaCl₂, 5 mM KC1, 0.3 mM KH₂PO₄,0.5 mM MgCl₂.6H₂), 0.4 mM MgSO₄.7H₂O, 138 mM NaCl, 4 mM NaHCO₃, 0.3 mMNaH₂PO₄.H₂O; 0.1X Hank's is made by mixing 1 parts 1X Hank's with 9parts PBS]. The resuspended virus pellet was incubated overnight at 4°C. without swirling. The virus pellet could be dispersed with gentlepipetting after the overnight incubation without significant loss ofinfectious virus. The titer of the virus stock was routinely increase100- to 300-fold after one round of ultracentrifugation. The efficiencyof recovery of infectious virus varied between 30 and 100%.

The virus stock was then subjected to low speed centrifugation in amicrofuge for 5 min at 4° C. to remove any visible cell debris oraggregated virions that were not resuspended under the above conditions(if the virus stock is not to be used for injection into oocytes orembryos, this centrifugation step may be omitted).

The virus stock was then subjected to another round ofultracentrifugation to concentrate the virus stock further. Theresuspended virus from the first round of centrifugation was pooled andpelleted by a second round of ultracentrifugation which was performed asdescribed above. Viral titers were increased approximately 2000-foldafter the second round of ultracentrifugation (titers of the pseudotypedLZRNL virus were typically greater than or equal to 1×10⁹ cfu/ml afterthe second round of ultracentrifugation).

The titers of the pre- and post-centrifugation fluids were determined byinfection of 208F (NIH 3T3 or Mac-T cells can also be employed) followedby selection of G418-resistant colonies as described above in Example 2.The concentrated viral stock was stable (i.e., did not lose infectivity)when stored at 4° C. for several weeks.

EXAMPLE 4

Preparation of Pseudotyped Retrovirus For Infection of Oocytes andEmbryos

The concentrated pseudotyped retrovirus were resuspended in 0.1X HBS(2.5 mM HEPES, pH 7.12, 14 mM NaCl, 75 μM Na₂HPO₄.H₂O) and 18 μlaliquots were placed in 0.5 ml vials (Eppendorf) and stored at −80° C.until used. The titer of the concentrated vector was determined bydiluting 1 μl of the concentrated virus 10⁻⁷- or 10⁻⁸ -fold with 0.1×HBS. The diluted virus solution was then used to infect 208F and Mac-Tcells and viral titers were determined as described in Example 2.

Prior to infection of oocytes or embryos (by microinjection), 1 μl ofpolybrene (25 ng/μl; the working solution of polybrene was generated bydiluting a stock solution having a concentration of 1 mg/ml [in sterileH₂O], in 0.1 HBS, pH 7.12) was mixed with 4 μl of concentrated virus toyield a solution containing 10³-10⁴ cfu/μl and 8 μg/ml polybrene. Thissolution was loaded into the injection needle (tip having an internaldiameter of approximately 2-4 μm) for injection into the perivitellinespace of gametes (pre-maturation oocytes, matured oocytes) or one cellstage zygotes (early stage embryo). An Eppendorf Transjector 5246 wasused for all microinjections.

EXAMPLE 5

Preparation and Microinjection of Gametes and Zygotes

Gametes (pre-maturation and pre-fertilization oocytes) and zygotes(fertilized oocytes) were prepared and microinjected with retroviralstocks as described below.

a) Solutions

Tyrodes-Lactate with HEPES (TL-HEPES): 114 mM NaCl, 3.2 mM KCI, 2.0 mMNaHCO₃, 0.4 mM Na₂H₂PO₄.H₂O, 10 mM Nalactate, 2 mM CaCl₂.2H₂O, 0.5 mMMgCl₂.6H₂O, 10 mM HEPES, 100 IU/ml penicillin, 50 μg/ml phenol red, Img/mI BSA fraction V, 0.2 mM pyruvate and 25 μg/ml gentamycin.

Maturation Medium: TC-199 medium (GIBCO) containing 10% FCS, 0.2 mMpyruvate, 5 μg/ml NIH o-LH (NIH), 25 μg/ml gentamycin and 1 μg/mlestradiol-17β.

Sperm-Tyrodes-Lactate (Sperm-TL): 100 mM NaCl, 3.2 mM KCI, 25 mM NaHCO₃,0.29 mM Na₂H₂PO₄.H₂O, 21.6 mM Na-lactate, 2.1 mM CaCl₂.2H₂O, 0.4 mMMgCl₂.6H₂O, 10 mM HEPES, 50 μg/ml phenol red, 6 mg/ml BSA fraction V,1.0 mM pyruvate and 25 μg/ml gentamycin.

Fertilization Medium: 114 mM NaCl, 3.2 mM KCI, 25 mM NaHCO₃, 0.4 mMNa₂H₂PO₄.H₂O, 10 mM Na-lactate, 2 mM CaCl₂.2H₂O, 0.5 mM MgCl₂.6H₂O, 100IU/ml penicillin, 50 μg/ml phenol red, 6 mg/mI BSA fatty acid free, 0.2mM pyruvate and 25 μg/ml gentamycin.

PHE: I mM hypotaurine, 2 mM penicillamine and 250 μM epinephrine.

Embryo Incubation+Amino Acids (EIAA): 114 μM NaCl, 3.2 μM KCI, 25 μMNaHCO₃, 1.6 μg/ml L(+)-lactate, 10.7 μg/ml L-glutamine, 300 μg/ml BSAfatty acid free, 0.275 μg/ml pyruvate, 25 μg/ml gentamycin, 10 μl of100× MEM amino acids stock (M7145, Sigma) per μl and 20 μl of 50× BMEamino acids stock (B6766, Sigma) per ml.

0.1× HBS: 2.5 mM HEPES (pH 7.12),14 mM NaCl and 75 μM Na₂HPO₄.H₂O.

b) Preparation, Injection, Maturation and

Fertilization of Pre-Maturation Oocytes

Oocytes were aspirated from small antral follicles on ovaries from dairycattle obtained from a slaughterhouse. Freshly aspirated oocytes at thegerminal vesicle (GV) stage, meiosis arrested, with the cumulus massattached were selected (i.e., pre-maturation oocytes). The oocytes werethen washed twice in freshly prepared TL-HEPES and transferred into a100 μl drop of TL-HEPES for microinjection.

Concentrated retroviral particles (prepared as described in Example 3)were resuspended in 0.1× HBS, mixed with polybrene and loaded into theinjection needle as described in Example 4. Approximately 10 pl of thevirus solution was then injected into the perivitelline space ofpre-maturation oocytes.

Following injection, the pre-maturation oocytes were washed twice infresh TL-HEPES and transferred into maturation medium (10 oocytes in 50μl). The pre-maturation oocytes were then incubated in Maturation Mediumfor 24 hours at 37° C. which permits the oocytes to mature to themetaphase 11 stage. The matured oocytes were then washed twice inSperm-TL and 10 oocytes were then transferred into 44 μl ofFertilization Medium. The mature oocytes (10 oocytes/44 μl FertilizationMedium) were then fertilized by the addition of 2 μl of sperm at aconcentration of 2.5×10⁷/ml, 2 μl of PHE and 2 μl of heparin(fertilization mixture). Sperm was prepared by discontinuous percollgradient separation of frozen-thawed semen as described (Kim et al.,Mot. Reprod. Develop., 35:105 [1993]). Briefly, percoll gradients wereformed by placing 2 ml of each of 90% and 45% percoll in a 15 ml conicaltube. Frozen-thawed semen was layered on top of the gradient and thetubes were centrifuged for 10 minutes at 700×g. Motile sperm werecollected from the bottom of the tube.

The oocytes were incubated for 16 to 24 hours at 37° C. in thefertilization mixture. Following fertilization, the cumulus cells wereremoved by vortexing the cells (one cell stage zygotes, PronucleusStage) for 3 minutes to produce “nude” oocytes. The nude oocytes werethen washed twice in embryo culture medium (ElAA) and 20 to 25 zygoteswere then cultured in 50 μl drop of ElAA (without serum until Day 4 atwhich time the zygotes were placed in EIAA containing 10% serum) untilthe desired developmental stage was reached: approximately 48 hours orDay 2 (Day 0 is the day when the matured oocytes are co-cultured withsperm) for morula stage (8 cell stage) or Day 6-7 for blastocyst stage.Embryos at the morula stage were analyzed for expression ofβ-galactosidase as described in Example 6. Embryos derived from injectedpre-maturation oocytes were also analyzed for β-galactosidase expressionat the 2 cell, 4 cell, and blastocyst stage and all developmental stagesexamined were positive.

c) Preparation, Injection and Fertilization of

Pre-Fertilization Oocytes

Pre-maturation oocytes were harvested, washed twice with TL-HEPES asdescribed above. The oocytes were then cultured in Maturation Medium (10oocytes per 50 μl medium) for 16 to 20 hours to producepre-fertilization oocytes (Metaphase 11 Stage). The pre-fertilization ormatured oocytes were then vortexed for 3 minutes to remove the cumuluscells to produce nude oocytes. The nude oocytes were washed twice inTL-HEPES and then transferred into a 100 μl drop of TL-HEPES formicroinjection. Microinjection was conducted as described above.

Following microinjection, the pre-fertilization oocytes were washedtwice with TL-HEPES and then placed in Maturation Medium untilfertilization. Fertilization was conducted as described above. Followingfertilization, the zygotes were then washed twice in ElAA and 20 to 25zygotes were then cultured per 50 μl drop of ElAA until the desireddevelopmental stage was reached. The embryos were then examined forP-galactosidase expression (Ex. 6) or transferred to recipient cows (Ex.7).

d) Preparation and Injection of One-Cell Stage Zygotes

Matured oocytes (Metaphase 11 stage) were generated as described above.The matured oocytes were then co-cultured in the presence of sperm for16 to 20 hours as described above to generate zygotes at the pronucleusstage. Zygotes at the pronucleus stage were vortexed for 3 minutes toremove the cumulus cell layer prior to microinjection. Microinjection ofretrovirus was conducted as described above. Following microinjection,the zygotes were washed four times in EIAA and then placed in an EIAAculture drop (25 zygotes per 50 μl drop of EIAA). The zygotes werecultured in EIAA (20 to 25 zygote per 50 μl drop of EIAA) until thedesired developmental stage was reached. The embryos were then examinedfor β-galactosidase expression (Ex. 6) or transferred to recipient cows(Ex. 7).

EXAMPLE 6

Injection of Pseudotyped Retrovirus Into the Perivitelline Space ofMaturing Bovine Oocytes Results in the Efficient Transfer of VectorSequences

Oocytes and one-cell zygotes which had been microirijected withpseudotyped LZRNL virus and cultured in vitro were examined forexpression of vector sequences by staining for β-galactosidase activitywhen the embryos had reached the morula stage. β-galactosidase activitywas examined as follows. Embryos were washed twice in PBS then fixed in0.5% glutaraldehyde in PBS containing 2 mM MgCl₂ for 40 min. at 40° C .The fixed embryos were then washed three times with PBS containing 2 mMMgCl₂ and then incubated at 37° C. overnight in X-gal solution (20 mMK₃Fe(CN)₆, 20 mM K₄Fe(CN)6.H₂O, 2 mM MgCl₂ and 1 mg/ml X-gal). Thepresence of a blue precipitate indicates expression of β-galactosidaseactivity. The results are shown in Table I below.

TABLE 1 % Positive For β-galactosidase Stage at Injection Stage atAnalysis Expression Pre-Fertilization Morula  47 (80/172)^(a) Oocyte(injected 20-24 hrs after exposure to Maturation Medium) Pronuclei StageMorula 25 (20/80) (injected 18-20 hrs after exposure to sperm) One-CellZygote Morula 25 (20/80) ^(a)Number positive/number injected.

From the results shown in Table 1, it is clear that infection ofpre-fertilization oocytes and zygotes using the methods of the presentinvention results in the transfer and expression of retrovirally encodednucleic acid. While not limiting the present invention to any particulartheory, it is currently believed that only half of the daughter cellsfrom an initial founder cell infected with a retrovirus will contain theprovirus because the retroviral provirus integrates intopost-replication host DNA (Hajihosseini et al., EMBO J., 12:4969[1993]). Therefore, the finding that 47% of the injectedpre-fertilization oocytes are positive for galactosidase expressionsuggests that 100% of these injected oocytes were infected with therecombinant retrovirus. Therefore, the methods of the present inventionprovide an efficiency of generating transgenic embryos which is superiorto existing methods.

EXAMPLE 7

Generation of Transgenic Cows Containing Integrated Retroviral NucleicAcid Sequences

Embryos derived from infected pre-fertilization oocytes and earlyzygotes were transferred into recipient cows which were allowed toprogress to term as described below.

a) Treatment of Embryos Derived From Infected Oocytes and Zygotes

Pre-fertilization oocytes (infected about 17 hours after exposure toMaturation Medium) and early stage zygotes (≧8 cell stage) were preparedand infected as described in Example 5 with the exceptions that 1) theVSV-G-pseudotyped virus used was the LSRNL virus which was prepared asdescribed for the LZRNL virus in Ex. 2, and 2) at day 4post-fertilization, embryos derived from injected pre-fertilizationoocytes and zygotes were placed in freshly prepared EIAA mediumcontaining 10% FCS and allowed to develop in vitro until transfer intorecipient cows. Embryos at Day 7 were transferred into recipient femaleswhich were prepared as described below.

b) Preparation of Recipient Cows and Embryo Transfer

Recipient cows were synchronized by injecting 100 μg ofgonadotropin-releasing hormone (GnRH; Sanofi Winthrop PharmaceuticalInc., New York, N.Y.) (Day 0). Seven days later, the recipients wereinjected with 25 mg of PGF2α(Upjohn Co., Kalamazoo, Miss.). Thirty to 48hours after injection of PGF2α, a second injection of 100 μg of GnRH wasgiven. Ovulation occurs about 24-32 hours post injection. Seven daysafter ovulation occurred, embryos derived from infected oocytes andzygotes (Day 7 embryos) were then transferred nonsurgically to the uterithe recipient cows. Two embryos were transferred into each recipient (itis expected that only one calf will be born from the transfer of twoembryos into a single recipient).

A total of 20 embryos were transferred into recipients on three separatedays. In the first transfer 8 embryos derived from infectedpre-fertilization oocytes were transferred into 4 recipients; fourcalves were born to these recipients and all four were found to bepositive for the presence of vector proviral DNA (i.e., 100% weretransgenic). In the second transfer, 8 embryos derived frompost-fertilization zygotes were transferred into 4 recipients; 2 calveswere born to these recipients and one of these animals was found to betransgenic (in the second transfer, one pregnancy was lost in the firstmonth and another pregnancy comprising twins was lost in the eighthmonth; neither embryo from the 8 month pregnancy was transgenic). In thethird transfer 4 embryos derived from infected zygotes (infected at the4-8 cell stage) were transferred into 2 recipients; 3 calves were bornto these recipients and none were transgenic.

The nine calves appeared healthy at birth and continue to appear healthyat the age of 6 months. Following the birth of offspring derived fromthe injected oocytes and zygotes, the offspring were examined bySouthern blot and PCR analyses to determine whether they contained theretroviral transgenes and whether they exhibited somatic cell mosaicism.Skin tissue and white blood cells (buffy coat) was collected from thecalves. Genomic DNA was extracted using standard techniques. Briefly,the tissue samples were digested with 50 μg/ml proteinase K (GIBCO) at55° C. The samples were then extracted sequentially twice with an equalvolume of phenol, once with phenol: chloroform (1:1) and once withchloroform. The DNA present in the aqueous layer was then precipitatedby the addition of 2 volumes of isopropanol. The DNA was collected bycentrifugation and the DNA pellet was resuspended in TE buffer (10 mMTris-Cl, 1 mM EDTA, pH 8.0) and the concentration was determinedspectrophotometrically. The DNA was then analyzed by Southern blottingand PCR analysis. The results are shown in FIGS. 2 and 3.

FIG. 2 shows an autoradiography of a Southern blot of genomic DNAisolated from the skin (FIG. 2A) and blood (FIG. 2B) of the six calvesderived from either pre-fertilization oocytes infected with VSVG-pseudotyped LSRNL virus at about 17 hours after exposure to MaturationMedium (calves numbered 17, 18, 20 and 21) or one cell zygotes infectedat about 12 hrs post-fertilization (calves numbered 15 and 16). The calfDNA was digested with HindIII which cuts the pLSRNL vector twice togenerate a 1.6 kb fragment (FIG. 2C). HindIII-digested DNA from theblood (lane labelled *12 derived from a random, nontransgenic calf),ovary and semen of nontransgenic cows (derived random adult females andmales) were also included. Lanes labeled “3989 M and F” represent DNAderived from two late term embryos that were born one month prematurely(these calves were generated from injected fertilized eggs and both arenontransgenic). Lanes labelled “LSRNL pDNA” contain HindIII-digestedpLSRNL plasmid DNA and provide controls for the quantitation of the copynumber of the integrated proviruses in the offspring (DNA equivalent to5, 10 or 25 copies of LSRNL were applied in these lanes).

Approximately 10 μg of the HindIll-digested DNAs were electrophoresed on0.8% agarose gels, and blotted onto a nylon membrane. The membrane washybridized with a ³²P labelled probe which hybridizes to the HBsAg genepresent in the pLZRNL vector (FIG. 2C). The HBsAG probe was generated byPCR amplification of pLSRNL plasmid DNA using the upstream primer S-1(5′-GGCTATCGCTGGATGTGTCT-3′; [SEQ ID NO:3]) and the downstream primerS-3 (5′-ACTGAACAAATGGCACTAGT-3′- [SEQ ID NO:4]). The PCR-generated probe(334 bp) was labeled using a Rediprime kit (Amersham, Arlington Heights,Ill.) according to the manufacturer's instructions. The autoradlographsshown in FIG. 2 were generated by exposure of the blots to X-ray filmfor 3 weeks at −80° C .

The results shown in FIG. 2 demonstrates that calves 16, 17, 18, 20 and21 contained retroviral vector DNA in both the skin (FIG. 2A) and blood(FIG. 2B). As blood cells (buffy coat) are derived from the mesoderm andskin cells are derived from the ectoderm, these results show that thetransgenic animals do not display somatic cell mosaicism. Southernblotting analysis has shown that the majority (i.e., 7/9) of thetransgenic calves contain a single copy of the proviral sequence; a few(i.e., 2/9) animals appear to contain two copies of the integratedproviral sequence. These results further demonstrate that retroviralinfection of both pre-fertilization oocytes and early stage zygotes wassuccessful in integrating the viral sequences into the genome of theresulting transgenic animals.

In order to confirm the presence of integrated retroviral sequences inthe genome of the transgenic animals' somatic cells, PCR analysis (FIG.3) was performed using genomic DNA isolated from the five transgeniccalves which were determined by Southern blot analysis to be transgenicfor the retroviral sequences. FIG. 3 shows the results of the PCRanalysis following amplification of two different regions (i.e., the neogene and the HbsAg gene) of the LZRNL retroviral genome which wasinjected into the oocytes. Genomic DNA from the skin and blood of eachof the five transgenic calves was amplified using the upstream anddownstream primers (SEQ ID NOS:1 and 2 and NOS:3 and 4; described supra)for the neo (FIG. 3A) and HBsAg (FIG. 3B) genes, respectively. The PCRswere conducted using the following thermocycling conditions: 94° C. ( 4min); (94° C. [2 min]; 50° C. [2 min]; 72° C. [2 min]) _(30 cycles); 72°C. (10 min). Amplification yielded the expected size of amplifiedsequence with the neo (349 bp) and HBsAg (334 bp) primers in both theblood and skin of each of the five transgenic calves. Genomic DNAisolated from the blood of non-transcyenic calves as well as from semenand ovary of non-transgenic cattle were used as negative controls in thePCRs. pLSRNL DNA was used as the positive control.

These data demonstrate that the infection of pre-fertilization oocytesresults in the efficient transfer of retroviral vector DNA (100% or 4transgenic calves/4 calves born from embryos derived from infectedpre-fertilization oocytes). In addition to providing a means forefficiently generating transgenic animals. The methods of the presentinvention provide a means for generating transgenic animals which do notdisplay somatic cell mosaicism. Further, these methods permit theproduction of transgenic animals which contain a single copy of thetransgene.

In order to confirm germ line transmission of the integrated viralsequences, the transgenic offspring are bred with non-transgenic cattleand the presence of the viral sequences (i.e., the transgene) determinedusing Southern blot analysis or PCR amplification as described above.Animals which are heterozygous or homozygous for the transgene areproduced using methods well known to the art (e.g., interbreeding ofanimals heterozygous for the transgene)

EXAMPLE 8

Detection of the HBsAg Transgene in the Sperm of Transgenic Bulls

Semen was collected from two transgenic bulls, #16 and #21. DNA wasisolated from the semen samples using methods known in the art. PCR wasthen conducted on the sample DNA, using the primers SI and S3, asdescribed below. The PCR results indicated that both bulls had thetransgene in their sperm.

These results demonstrated that transgenic bulls produced either byperivitelline space injection of an unfertilized oocyte (#21) or byperivitelline space injection of a fertilized zygote (#16) have thetransgene present in their sperm, and are thus capable of passing thetransgene on to their offspring. Indeed, as described in Example 9below, bull #16 has produced two live transgenic offspring.

EXAMPLE 9

Confirmation of Transgene Stability

To confirm the transgene stability of a transgenic bull produced asdescribed in the previous Examples, and to determine whether the genewas behaving in a normal Mendelian fashion, a transgenic bull(designated as #16) produced through one-cell zygotic injection, wasnaturally mated with a non-transgenic cow. This mating resulted in theproduction of twill calves, one female (designated as #42) and one male(designated as #43). Blood and skin samples were taken from each of thecalves, and their DNA was isolated using methods known in the art (Seee.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. [1986]). PCR wasperformed on these DNA samples, using the methods described in Example7, above. Two sets of primers were used to analyze both the blood andskin samples. One set of primers (“Neo1” and “Neo2”) was used to detectthe neomycin resistance gene in the LSRNL vector. The location anddescription of these primers is shown in FIG. 3A. The second set ofprimers (S1 and S3) were used to detect a portion of the Hepatitis Bsurface antigen (HbsAg) gene in the LSRNL vector. The location anddescription of these primers is shown in FIG. 3B. Both the skin andblood samples from these calves were positive for the LSRNL transgene,indicating that the gene can be transmitted from an original transgenicbull created by one-cell zygotic injection, to his offspring. FIG. 4shows the results of PCR screening of skin samples from these calves. Inthis gel, the control animal is indicated as #12, while the offspringare indicated as #42 and #43, as described above. Lane one contains DNAsize standards, and lanes 2-4 contain the DNA samples analyzed using theneo PCR primers, while lanes 5-7 contain the same DNA samples analyzedusing the HBsAg PCR primers. The correct size for the neo band is 349base pairs, while the correct size for the HBsAg band is slightlysmaller, at 334 base pairs.

These data demonstrate that transgenic animals can be successfullycreated by perivitelline space injection of a one-cell zygote with apseudotyped replication-defective retrovirus. In addition, these dataalso demonstrate that the incorporated transgene is passed on theoffspring of the transgenic animal.

EXAMPLE 10

Production of HBsAg in Milk of Transgenic Cows

In this experiment, female founder transgenic heifers (designated as #17and #18), were artificially induced to lactate at 22 months of age,using a protocol described by Dommer (Dommer, “Artificial Induction ofLactation in Nulligravida Heifers,” MS Thesis, University of Wisconsin,Madison, 1996; and Dommer and Bremel, J. Dairy Sci., 79 (Suppl. 1):146[1996]). After induction of lactation and the subsequent secretion ofmilk, the milk was assayed for the presence of HBsAg.

Milk samples were collected from #17 and #918, and five control heifersthat had also been artificially induced to lactate using the sameprotocol and at the same time as #17 and #18. Whole milk samples wereanalyzed using the AUSZYME® Monoclonal Antibody Assay (Abbott), for thedetection of HbsAg.

The milk samples collected from #17 and #18 tested positive for HBsAg.The milk samples from the five control heifers were all negative for theantigen. The estimated level of HBsAg production, based on the AUSZYME®kit and its positive control, as well as a dilution series of the milksamples, was found to be 200 ng HBsAg/mI milk, for #17, and 700 ngHBsAg/ml milk, for #18.

These data clearly demonstrate that transgenic animals produced byperivitelline space injection of an unfertilized oocyte are capable ofproducing substantial levels of foreign proteins in their milk. Inaddition, these experiments also demonstrate the utility of using theMoMLV LTR as a promoter for driving the production of foreign proteinsin the milk of transgenic cattle, as this promoter was shown to becapable of causing the production of HBsAg in the milk of thesetransgenic animals. In addition, the expression of an exonless construct(i.e., with the LTR of the present invention) indicates that the LTR isalso functioning as an enhancer. Furthermore, these data clearly showthat the expression system of the present invention is capable ofpreferential mammary expression, even tliougli the MoMLV LTR is not a“mammary-specific” promoter.

EXAMPLE 11

Presence HBsAg in the Serum and Urine of Transgenic Cattle

In addition to milk samples, blood and urine samples were also collectedfrom the two female founder transgenic heifers #17 and #18. The serumwas separated from the whole blood using methods known in the art (i.e.,centrifugation). The urine and serum samples were assayed for thepresence of HBsAg using the AUSZYME® system, as per the kitmanufacturer's instructions. The urine and serum of #17 and #18 alltested positive for the presence of HBsAg, while the urine and serumsamples from the control animals all tested negative. Based on this testsystem, the estimated level of HBsAg production for #17 was 2.58 ngHBsAg/ml of serum, and 0.64 ng HBsAg/ml of urine. For #18, the valueswere 0.64 ng HBsAg/ml of serum, and 0.97 ng HBsAg/ml of urine.

These data demonstrate that transgenic animals produced by perivitellinespace injection of an unfertilized oocyte are capable of producingsubstantial levels of foreign proteins in their serum and urine. Thesedata further demonstrate the utility of using the MoMLV LTR as apromoter for driving the constitutive production of foreign proteins intransgenic cattle, as this promoter was shown in these experiments tocause the production of HBsAg in milk, serum, and urine of transgeniccattle. As used herein, the term “constitutive” refers to a relativelylow level of expression throughout the animal's body. In contrast, theterm “preferentially expressed” indicates that a relatively high levelof expression is achieved in certain tissues or body fluids, as comparedto other tissues and fluids. For example, in preferred embodiments ofthe present invention, foreign proteins of interest are preferentiallyexpressed in such fluids as milk.

It is contemplated that such a promoter could be used to controlexpression of proteins that would prevent disease and/or infection inthe transgenic animals and their offspring, or be of use in theproduction of a consistent level of protein expression in a number ofdifferent tissues and body fluids.

From the above it is clear that the invention provides improved methodsand compositions for the production of transgenic non-human animals. Themethods of the present invention provide for the production oftransgenic non-human animals with improved efficiency and a reducedincidence of generating animals which are mosaic for the presence of thetransgene.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, transgenic animals, or related fields are intended tobe within the scope of the following claims.

10 20 base pairs nucleic acid single linear other nucleic acid /desc =“DNA” not provided 1 GCATTGCATC AGCCATGATG 20 20 base pairs nucleic acidsingle linear other nucleic acid /desc = “DNA” not provided 2 GATGGATTGCACGCAGGTTC 20 20 base pairs nucleic acid single linear other nucleicacid /desc = “DNA” not provided 3 GGCTATCGCT GGATGTGTCT 20 20 base pairsnucleic acid single linear other nucleic acid /desc = “DNA” not provided4 ACTGAACAAA TGGCACTAGT 20 1590 base pairs nucleic acid double linearother nucleic acid /desc = “DNA” not provided CDS 1..1587 5 ATG GAT CTCTTT CCC ATT TTG GTC GTG GTG CTC ATG ACA GAT ACT GTC 48 Met Asp Leu PhePro Ile Leu Val Val Val Leu Met Thr Asp Thr Val 1 5 10 15 TTA GGG AAGTTT CAA ATT GTC TTC CCG GAT CAG AAT GAA CTG GAG TGG 96 Leu Gly Lys PheGln Ile Val Phe Pro Asp Gln Asn Glu Leu Glu Trp 20 25 30 AGA CCA GTT GTGGGT GAC TCT CGG CAT TGC CCA CAG TCA TCA GAA ATG 144 Arg Pro Val Val GlyAsp Ser Arg His Cys Pro Gln Ser Ser Glu Met 35 40 45 CAA TTC GAT GGA AGCAGA TCC CAG ACC ATA CTG ACT GGG AAA GCT CCC 192 Gln Phe Asp Gly Ser ArgSer Gln Thr Ile Leu Thr Gly Lys Ala Pro 50 55 60 GTG GGG ATC ACG CCC TCTAAA TCA GAT GGA TTT ATC TGC CAT GCC GCA 240 Val Gly Ile Thr Pro Ser LysSer Asp Gly Phe Ile Cys His Ala Ala 65 70 75 80 AAA TGG GTG ACA ACA TGTGAT TTC AGG TGG TAT GGG CCG AAA TAC ATC 288 Lys Trp Val Thr Thr Cys AspPhe Arg Trp Tyr Gly Pro Lys Tyr Ile 85 90 95 ACT CAT TCA ATA CAT CAT CTGAGA CCG ACA ACA TCA GAC TGT GAG ACA 336 Thr His Ser Ile His His Leu ArgPro Thr Thr Ser Asp Cys Glu Thr 100 105 110 GCT CTC CAA AGG TAT AAA GATGGG AGC TTA ATC AAT CTT GGA TTC CCC 384 Ala Leu Gln Arg Tyr Lys Asp GlySer Leu Ile Asn Leu Gly Phe Pro 115 120 125 CCA GAA TCC TGC GGT TAT GCAACA GTC ACA GAT TCT GAG GCA ATG TTG 432 Pro Glu Ser Cys Gly Tyr Ala ThrVal Thr Asp Ser Glu Ala Met Leu 130 135 140 GTC CAA GTG ACT CCC CAC CACGTT GGG GTG GAT GAT TAT AGA GGT CAC 480 Val Gln Val Thr Pro His His ValGly Val Asp Asp Tyr Arg Gly His 145 150 155 160 TGG ATC GAC CCA CTA TTTCCA GGA GGA GAA TGC TCC ACC AAT TTT TGT 528 Trp Ile Asp Pro Leu Phe ProGly Gly Glu Cys Ser Thr Asn Phe Cys 165 170 175 GAT ACA GTC CAC AAT TCATCG GTG TGG ATC CCC AAG AGT CAA AAG ACT 576 Asp Thr Val His Asn Ser SerVal Trp Ile Pro Lys Ser Gln Lys Thr 180 185 190 GAC ATC TGT GCC CAG TCTTTC AAA AAT ATC AAG ATG ACC GCA TCT TAC 624 Asp Ile Cys Ala Gln Ser PheLys Asn Ile Lys Met Thr Ala Ser Tyr 195 200 205 CCC TCA GAA GGA GCA TTGGTG AGT GAC AGA TTT GCC TTC CAC AGT GCA 672 Pro Ser Glu Gly Ala Leu ValSer Asp Arg Phe Ala Phe His Ser Ala 210 215 220 TAT CAT CCA AAT ATG CCGGGG TCA ACT GTT TGC ATA ATG GAC TTT TGC 720 Tyr His Pro Asn Met Pro GlySer Thr Val Cys Ile Met Asp Phe Cys 225 230 235 240 GAA CAA AAG GGG TTGAGA TTC ACA AAT GGA GAG TGG ATG GGT CTC AAT 768 Glu Gln Lys Gly Leu ArgPhe Thr Asn Gly Glu Trp Met Gly Leu Asn 245 250 255 GTG GAG CAA TCC ATCCGA GAG AAG AAG ATA AGT GCC ATC TTC CCA AAT 816 Val Glu Gln Ser Ile ArgGlu Lys Lys Ile Ser Ala Ile Phe Pro Asn 260 265 270 TGT GTT GCA GGG ACTGAA ATC CGA GCC ACA CTA GAA TCA GAA GGG GCA 864 Cys Val Ala Gly Thr GluIle Arg Ala Thr Leu Glu Ser Glu Gly Ala 275 280 285 AGA ACT TTG ACG TGGGAG ACT CAA AGA ATG CTA GAT TAC TCT TTG TGT 912 Arg Thr Leu Thr Trp GluThr Gln Arg Met Leu Asp Tyr Ser Leu Cys 290 295 300 CAG AAC ACC TGG GACAAA GTT TCC AGG AAA GAA CCT CTC AGT CCG CTT 960 Gln Asn Thr Trp Asp LysVal Ser Arg Lys Glu Pro Leu Ser Pro Leu 305 310 315 320 GAC TTG AGC TATCTG TCA CCA AGG GCT CCA GGG AAA GGC ATG GCC TAT 1008 Asp Leu Ser Tyr LeuSer Pro Arg Ala Pro Gly Lys Gly Met Ala Tyr 325 330 335 ACC GTC ATA AACGGA ACC CTG CAT TCG GCT CAT GCT AAA TAC ATT AGA 1056 Thr Val Ile Asn GlyThr Leu His Ser Ala His Ala Lys Tyr Ile Arg 340 345 350 ACC TGG ATT GATTAT GGA GAA ATG AAG GAA ATT AAA GGT GGA CGT GGA 1104 Thr Trp Ile Asp TyrGly Glu Met Lys Glu Ile Lys Gly Gly Arg Gly 355 360 365 GAA TAT TCC AAGGCT CCT GAG CTC CTC TGG TCC CAG TGG TTC GAT TTT 1152 Glu Tyr Ser Lys AlaPro Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe 370 375 380 GGA CCG TTC AAAATT GGA CCG AAT GGA CTC CTG CAC ACA GGG AAA ACC 1200 Gly Pro Phe Lys IleGly Pro Asn Gly Leu Leu His Thr Gly Lys Thr 385 390 395 400 TTT AAA TTCCCT CTT TAT TTG ATC GGA GCA GGC ATA ATT GAC GAA GAT 1248 Phe Lys Phe ProLeu Tyr Leu Ile Gly Ala Gly Ile Ile Asp Glu Asp 405 410 415 CTG CAT GAACTA GAT GAG GCT GCT CCC ATT GAT CAC CCA CAA ATG CCT 1296 Leu His Glu LeuAsp Glu Ala Ala Pro Ile Asp His Pro Gln Met Pro 420 425 430 GAC GCG AAAAGC GTT CTT CCA GAA GAT GAA GAG ATA TTC TTC GGA GAC 1344 Asp Ala Lys SerVal Leu Pro Glu Asp Glu Glu Ile Phe Phe Gly Asp 435 440 445 ACA GGT GTATCC AAA AAC CCT ATC GAG TTG ATT CAA GGA TGG TTC TCA 1392 Thr Gly Val SerLys Asn Pro Ile Glu Leu Ile Gln Gly Trp Phe Ser 450 455 460 AAT TGG AGAGAG AGT GTA ATG GCA ATA GTC GGA ATT GTT CTA CTC ATC 1440 Asn Trp Arg GluSer Val Met Ala Ile Val Gly Ile Val Leu Leu Ile 465 470 475 480 GTT GTGACA TTT CTG GCG ATC AAG ACG GTC CGG GTG CTT AAT TGT CTC 1488 Val Val ThrPhe Leu Ala Ile Lys Thr Val Arg Val Leu Asn Cys Leu 485 490 495 TGG AGACCC AGA AAG AAA AGA ATC GTC AGA CAA GAA GTA GAT GTT GAA 1536 Trp Arg ProArg Lys Lys Arg Ile Val Arg Gln Glu Val Asp Val Glu 500 505 510 TCC CGACTA AAC CAT TTT GAG ATG AGA GGC TTT CCT GAA TAT GTT AAG 1584 Ser Arg LeuAsn His Phe Glu Met Arg Gly Phe Pro Glu Tyr Val Lys 515 520 525 AGA TAA1590 Arg 529 amino acids amino acid linear protein not provided 6 MetAsp Leu Phe Pro Ile Leu Val Val Val Leu Met Thr Asp Thr Val 1 5 10 15Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln Asn Glu Leu Glu Trp 20 25 30Arg Pro Val Val Gly Asp Ser Arg His Cys Pro Gln Ser Ser Glu Met 35 40 45Gln Phe Asp Gly Ser Arg Ser Gln Thr Ile Leu Thr Gly Lys Ala Pro 50 55 60Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe Ile Cys His Ala Ala 65 70 7580 Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile 85 9095 Thr His Ser Ile His His Leu Arg Pro Thr Thr Ser Asp Cys Glu Thr 100105 110 Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu Ile Asn Leu Gly Phe Pro115 120 125 Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp Ser Glu Ala MetLeu 130 135 140 Val Gln Val Thr Pro His His Val Gly Val Asp Asp Tyr ArgGly His 145 150 155 160 Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys SerThr Asn Phe Cys 165 170 175 Asp Thr Val His Asn Ser Ser Val Trp Ile ProLys Ser Gln Lys Thr 180 185 190 Asp Ile Cys Ala Gln Ser Phe Lys Asn IleLys Met Thr Ala Ser Tyr 195 200 205 Pro Ser Glu Gly Ala Leu Val Ser AspArg Phe Ala Phe His Ser Ala 210 215 220 Tyr His Pro Asn Met Pro Gly SerThr Val Cys Ile Met Asp Phe Cys 225 230 235 240 Glu Gln Lys Gly Leu ArgPhe Thr Asn Gly Glu Trp Met Gly Leu Asn 245 250 255 Val Glu Gln Ser IleArg Glu Lys Lys Ile Ser Ala Ile Phe Pro Asn 260 265 270 Cys Val Ala GlyThr Glu Ile Arg Ala Thr Leu Glu Ser Glu Gly Ala 275 280 285 Arg Thr LeuThr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys 290 295 300 Gln AsnThr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser Pro Leu 305 310 315 320Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys Gly Met Ala Tyr 325 330335 Thr Val Ile Asn Gly Thr Leu His Ser Ala His Ala Lys Tyr Ile Arg 340345 350 Thr Trp Ile Asp Tyr Gly Glu Met Lys Glu Ile Lys Gly Gly Arg Gly355 360 365 Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser Gln Trp Phe AspPhe 370 375 380 Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu His Thr GlyLys Thr 385 390 395 400 Phe Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly IleIle Asp Glu Asp 405 410 415 Leu His Glu Leu Asp Glu Ala Ala Pro Ile AspHis Pro Gln Met Pro 420 425 430 Asp Ala Lys Ser Val Leu Pro Glu Asp GluGlu Ile Phe Phe Gly Asp 435 440 445 Thr Gly Val Ser Lys Asn Pro Ile GluLeu Ile Gln Gly Trp Phe Ser 450 455 460 Asn Trp Arg Glu Ser Val Met AlaIle Val Gly Ile Val Leu Leu Ile 465 470 475 480 Val Val Thr Phe Leu AlaIle Lys Thr Val Arg Val Leu Asn Cys Leu 485 490 495 Trp Arg Pro Arg LysLys Arg Ile Val Arg Gln Glu Val Asp Val Glu 500 505 510 Ser Arg Leu AsnHis Phe Glu Met Arg Gly Phe Pro Glu Tyr Val Lys 515 520 525 Arg 1590base pairs nucleic acid double linear other nucleic acid /desc = “DNA”not provided CDS 1..1587 7 ATG GAT CTC TTT CCC ATT TTG GTC GTG GTG CTCATG ACA GAT ACT GTC 48 Met Asp Leu Phe Pro Ile Leu Val Val Val Leu MetThr Asp Thr Val 1 5 10 15 TTA GGG AAG TTT CAA ATT GTC TTC CCG GAT CAGAAT GAA CTG GAG TGG 96 Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln AsnGlu Leu Glu Trp 20 25 30 AGA CCA GTT GTG GGT GAC TCT CGG CAT TGC CCA CAGTCA TCA GAA ATG 144 Arg Pro Val Val Gly Asp Ser Arg His Cys Pro Gln SerSer Glu Met 35 40 45 CAA TTC GAT GGA AGC AGA TCC CAG ACC ATA CTG ACT GGGAAA GCT CCC 192 Gln Phe Asp Gly Ser Arg Ser Gln Thr Ile Leu Thr Gly LysAla Pro 50 55 60 GTG GGG ATC ACG CCC TCT AAA TCA GAT GGA TTT ATC TGC CATGCC GCA 240 Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe Ile Cys His AlaAla 65 70 75 80 AAA TGG GTG ACA ACA TGT GAT TTC AGG TGG TAT GGG CCG AAATAC ATC 288 Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys TyrIle 85 90 95 ACT CAT TCA ATA CAT CAT CTG AGA CCG ACA ACA TCA GAC TGT GAGACA 336 Thr His Ser Ile His His Leu Arg Pro Thr Thr Ser Asp Cys Glu Thr100 105 110 GCT CTC CAA AGG TAT AAA GAT GGG AGC TTA ATC AAT CTT GGA TTCCCC 384 Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu Ile Asn Leu Gly Phe Pro115 120 125 CCA GAA TCC TGC GGT TAT GCA ACA GTC ACA GAT TCT GAG GCA ATGTTG 432 Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp Ser Glu Ala Met Leu130 135 140 GTC CAA GTG ACT CCC CAC CAC GTT GGG GTG GAT GAT TAT AGA GGTCAC 480 Val Gln Val Thr Pro His His Val Gly Val Asp Asp Tyr Arg Gly His145 150 155 160 TGG ATC GAC CCA CTA TTT CCA GGA GGA GAA TGC TCC ACC AATTTT TGT 528 Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn PheCys 165 170 175 GAT ACA GTC CAC AAT TCA TCG GTG TGG ATC CCC AAG AGT CAAAAG ACT 576 Asp Thr Val His Asn Ser Ser Val Trp Ile Pro Lys Ser Gln LysThr 180 185 190 GAC ATC TGT GCC CAG TCT TTC AAA AAT ATC AAG ATG ACC GCATCT TAC 624 Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys Met Thr Ala SerTyr 195 200 205 CCC TCA GAA GGA GCA TTG GTG AGT GAC AGA TTT GCC TTC CACAGT GCA 672 Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Ala Phe His SerAla 210 215 220 TAT CAT CCA AAT ATG CCG GGG TCA ACT GTT TGC ATA ATG GACTTT TGC 720 Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys Ile Met Asp PheCys 225 230 235 240 GAA CAA AAG GGG TTG AGA TTC ACA AAT GGA GAG TGG ATGGGT CTC AAT 768 Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met GlyLeu Asn 245 250 255 GTG GAG CAA TCC ATC CGA GAG AAG AAG ATA AGT GCC ATCTTC CCA AAT 816 Val Glu Gln Ser Ile Arg Glu Lys Lys Ile Ser Ala Ile PhePro Asn 260 265 270 TGT GTT GCA GGG ACT GAA ATC CGA GCC ACA CTA GAA TCAGAA GGG GCA 864 Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu Glu Ser GluGly Ala 275 280 285 AGA ACT TTG ACG TGG GAG ACT CAA AGA ATG CTA GAT TACTCT TTG TGT 912 Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr SerLeu Cys 290 295 300 CAG AAC ACC TGG GAC AAA GTT TCC AGG AAA GAA CCT CTCAGT CCG CTT 960 Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu SerPro Leu 305 310 315 320 GAC TTG AGC TAT CTG TCA CCA AGG GCT CCA GGG AAAGGC ATG GCC TAT 1008 Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys GlyMet Ala Tyr 325 330 335 ACC GTC ATA AAC GGA ACC CTG CAT TCG GCT CAT GCTAAA TAC ATT AGA 1056 Thr Val Ile Asn Gly Thr Leu His Ser Ala His Ala LysTyr Ile Arg 340 345 350 ACC TGG ATT GAT TAT GGA GAA ATG AAG GAA ATT AAAGGT GGA CGT GGA 1104 Thr Trp Ile Asp Tyr Gly Glu Met Lys Glu Ile Lys GlyGly Arg Gly 355 360 365 GAA TAT TCC AAG GCT CCT GAG CTC CTC TGG TCC CAGTGG TTC GAT TTT 1152 Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser Gln TrpPhe Asp Phe 370 375 380 GGA CCG TTC AAA ATT GGA CCG AAT GGA CTC CTG CACACA GGG AAA ACC 1200 Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu His ThrGly Lys Thr 385 390 395 400 TTT AAA TTC CCT CTT TAT TTG ATC GGA GCA GGCATA ATT GAC GAA GAT 1248 Phe Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly IleIle Asp Glu Asp 405 410 415 CTG CAT GAA CTA GAT GAG GCT GCT CCC ATT GATCAC CCA CAA ATG CCT 1296 Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp HisPro Gln Met Pro 420 425 430 GAC GCG AAA AGC GTT CTT CCA GAA GAT GAA GAGATA TTC TTC GGA GAC 1344 Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu IlePhe Phe Gly Asp 435 440 445 ACA GGT GTA TCC AAA AAC CCT ATC GAG TTG ATTCAA GGA TGG TTC TCA 1392 Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile GlnGly Trp Phe Ser 450 455 460 AAT TGG AGA GAG AGT GTA ATG GCA ATA GTC GGAATT GTT CTA CTC ATC 1440 Asn Trp Arg Glu Ser Val Met Ala Ile Val Gly IleVal Leu Leu Ile 465 470 475 480 GTT GTG ACA TTT CTG GCG ATC AAG ACG GTCCGG GTG CTT AAT TGT CTC 1488 Val Val Thr Phe Leu Ala Ile Lys Thr Val ArgVal Leu Asn Cys Leu 485 490 495 TGG AGA CCC AGA AAG AAA AGA ATC GTC AGACAA GAA GTA GAT GTT GAA 1536 Trp Arg Pro Arg Lys Lys Arg Ile Val Arg GlnGlu Val Asp Val Glu 500 505 510 TCC CGA CTA AAC CAT TTT GAG ATG AGA GGCTTT CCT GAA TAT GTT AAG 1584 Ser Arg Leu Asn His Phe Glu Met Arg Gly PhePro Glu Tyr Val Lys 515 520 525 AGA TAA 1590 Arg 529 amino acids aminoacid linear protein not provided 8 Met Asp Leu Phe Pro Ile Leu Val ValVal Leu Met Thr Asp Thr Val 1 5 10 15 Leu Gly Lys Phe Gln Ile Val PhePro Asp Gln Asn Glu Leu Glu Trp 20 25 30 Arg Pro Val Val Gly Asp Ser ArgHis Cys Pro Gln Ser Ser Glu Met 35 40 45 Gln Phe Asp Gly Ser Arg Ser GlnThr Ile Leu Thr Gly Lys Ala Pro 50 55 60 Val Gly Ile Thr Pro Ser Lys SerAsp Gly Phe Ile Cys His Ala Ala 65 70 75 80 Lys Trp Val Thr Thr Cys AspPhe Arg Trp Tyr Gly Pro Lys Tyr Ile 85 90 95 Thr His Ser Ile His His LeuArg Pro Thr Thr Ser Asp Cys Glu Thr 100 105 110 Ala Leu Gln Arg Tyr LysAsp Gly Ser Leu Ile Asn Leu Gly Phe Pro 115 120 125 Pro Glu Ser Cys GlyTyr Ala Thr Val Thr Asp Ser Glu Ala Met Leu 130 135 140 Val Gln Val ThrPro His His Val Gly Val Asp Asp Tyr Arg Gly His 145 150 155 160 Trp IleAsp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys 165 170 175 AspThr Val His Asn Ser Ser Val Trp Ile Pro Lys Ser Gln Lys Thr 180 185 190Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys Met Thr Ala Ser Tyr 195 200205 Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Ala Phe His Ser Ala 210215 220 Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys Ile Met Asp Phe Cys225 230 235 240 Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met GlyLeu Asn 245 250 255 Val Glu Gln Ser Ile Arg Glu Lys Lys Ile Ser Ala IlePhe Pro Asn 260 265 270 Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu GluSer Glu Gly Ala 275 280 285 Arg Thr Leu Thr Trp Glu Thr Gln Arg Met LeuAsp Tyr Ser Leu Cys 290 295 300 Gln Asn Thr Trp Asp Lys Val Ser Arg LysGlu Pro Leu Ser Pro Leu 305 310 315 320 Asp Leu Ser Tyr Leu Ser Pro ArgAla Pro Gly Lys Gly Met Ala Tyr 325 330 335 Thr Val Ile Asn Gly Thr LeuHis Ser Ala His Ala Lys Tyr Ile Arg 340 345 350 Thr Trp Ile Asp Tyr GlyGlu Met Lys Glu Ile Lys Gly Gly Arg Gly 355 360 365 Glu Tyr Ser Lys AlaPro Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe 370 375 380 Gly Pro Phe LysIle Gly Pro Asn Gly Leu Leu His Thr Gly Lys Thr 385 390 395 400 Phe LysPhe Pro Leu Tyr Leu Ile Gly Ala Gly Ile Ile Asp Glu Asp 405 410 415 LeuHis Glu Leu Asp Glu Ala Ala Pro Ile Asp His Pro Gln Met Pro 420 425 430Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu Ile Phe Phe Gly Asp 435 440445 Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile Gln Gly Trp Phe Ser 450455 460 Asn Trp Arg Glu Ser Val Met Ala Ile Val Gly Ile Val Leu Leu Ile465 470 475 480 Val Val Thr Phe Leu Ala Ile Lys Thr Val Arg Val Leu AsnCys Leu 485 490 495 Trp Arg Pro Arg Lys Lys Arg Ile Val Arg Gln Glu ValAsp Val Glu 500 505 510 Ser Arg Leu Asn His Phe Glu Met Arg Gly Phe ProGlu Tyr Val Lys 515 520 525 Arg 1569 base pairs nucleic acid doublelinear other nucleic acid /desc = “DNA” not provided CDS 1..1566 9 ATGAAT ATA CCT TGC TTT GCT GTG ATC CTC AGC TTA GCT ACT ACA CAT 48 Met AsnIle Pro Cys Phe Ala Val Ile Leu Ser Leu Ala Thr Thr His 1 5 10 15 TCTCTG GGA GAA TTC CCC TTG TAT ACG ATT CCC GAG AAA ATA GAG AAA 96 Ser LeuGly Glu Phe Pro Leu Tyr Thr Ile Pro Glu Lys Ile Glu Lys 20 25 30 TGG ACCCCC ATA GAC ATG ATC CAT CTT AGT TGC CCT AAT AAC ATG CTG 144 Trp Thr ProIle Asp Met Ile His Leu Ser Cys Pro Asn Asn Met Leu 35 40 45 TCT GAG GAAGAA GGT TGC AAT ACA GAG TCT CCT TTC ACC TAC TTC GAG 192 Ser Glu Glu GluGly Cys Asn Thr Glu Ser Pro Phe Thr Tyr Phe Glu 50 55 60 CTC AAG AGT GGTTAC CTA GCC CAT CAG AAG GTC CCA GGA TTT ACA TGC 240 Leu Lys Ser Gly TyrLeu Ala His Gln Lys Val Pro Gly Phe Thr Cys 65 70 75 80 ACT GGG GTT GTGAAT GAG GCA GAG ACA TAC ACA AAC TTT GTC GGA TAT 288 Thr Gly Val Val AsnGlu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95 GTC ACC ACC ACC TTCAAA AGG AAG CAC TTT AAA CCT ACA GTG GCT GCT 336 Val Thr Thr Thr Phe LysArg Lys His Phe Lys Pro Thr Val Ala Ala 100 105 110 TGT CGT GAT GCC TACAAC TGG AAA GTA TCA GGG GAC CCC CGA TAT GAA 384 Cys Arg Asp Ala Tyr AsnTrp Lys Val Ser Gly Asp Pro Arg Tyr Glu 115 120 125 GAA TCT CTA CAC ACCCCG TAT CCC GAC AGC AGC TGG TTA AGG ACT GTG 432 Glu Ser Leu His Thr ProTyr Pro Asp Ser Ser Trp Leu Arg Thr Val 130 135 140 ACC ACA ACC AAA GAAGCC CTT CTT ATA ATA TCG CCA AGC ATT GTA GAG 480 Thr Thr Thr Lys Glu AlaLeu Leu Ile Ile Ser Pro Ser Ile Val Glu 145 150 155 160 ATG GAC ATA TATGGC AGG ACC CTT CAC TCT CCC ATG TTC CCT TCG GGG 528 Met Asp Ile Tyr GlyArg Thr Leu His Ser Pro Met Phe Pro Ser Gly 165 170 175 AAA TGT TCC AAGCTC TAT CCT TCT GTC CCC TCT TGT ACA ACC AAC CAT 576 Lys Cys Ser Lys LeuTyr Pro Ser Val Pro Ser Cys Thr Thr Asn His 180 185 190 GAT TAC ACA TTGTGG TTG CCA GAA GAT TCT AGT CTG AGT TTG ATT TGC 624 Asp Tyr Thr Leu TrpLeu Pro Glu Asp Ser Ser Leu Ser Leu Ile Cys 195 200 205 GAC ATC TTC ACTTCC AGC AGT GGA CAG AAG GCC ATG AAT GGG TCT CGC 672 Asp Ile Phe Thr SerSer Ser Gly Gln Lys Ala Met Asn Gly Ser Arg 210 215 220 ATC TGC GGA TTCAAG GAT GAA AGG GGA TTT TAC AGA TCC TTG AAG GGA 720 Ile Cys Gly Phe LysAsp Glu Arg Gly Phe Tyr Arg Ser Leu Lys Gly 225 230 235 240 TCC TGT AAGCTG ACA TTG TGC GGG AAA CCT GGA ATT AGG CTG TTC GAC 768 Ser Cys Lys LeuThr Leu Cys Gly Lys Pro Gly Ile Arg Leu Phe Asp 245 250 255 GGA ACT TGGGTC TCT TTT ACA AAG CCG GAC GTT CAT GTG TGG TGC ACT 816 Gly Thr Trp ValSer Phe Thr Lys Pro Asp Val His Val Trp Cys Thr 260 265 270 CCC AAC CAGTTA GTC AAT ATA CAT AAC GAC AGA CTA GAT GAG GTT GAA 864 Pro Asn Gln LeuVal Asn Ile His Asn Asp Arg Leu Asp Glu Val Glu 275 280 285 CAT CTG ATCGTG GAC GAT ATC ATC AAG AAG AGA GAG GAG TGT TTA GAC 912 His Leu Ile ValAsp Asp Ile Ile Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300 ACG CTG GAAACT ATA CTT ATG TCT CAA TCA GTT AGT TTT AGA CGG TTG 960 Thr Leu Glu ThrIle Leu Met Ser Gln Ser Val Ser Phe Arg Arg Leu 305 310 315 320 AGC CATTTC AGA AAG TTA GTT CCA GGA TAT GGA AAA GCT TAC ACT ATT 1008 Ser His PheArg Lys Leu Val Pro Gly Tyr Gly Lys Ala Tyr Thr Ile 325 330 335 TTG AACGGC AGC TTA ATG GAA ACA AAT GTC TAC TAC AAA AGA GTT GAC 1056 Leu Asn GlySer Leu Met Glu Thr Asn Val Tyr Tyr Lys Arg Val Asp 340 345 350 AGG TGGGCG GAC ATT TTG CCT TCT AGG GGA TGT CTG AAA GTC GGA CAA 1104 Arg Trp AlaAsp Ile Leu Pro Ser Arg Gly Cys Leu Lys Val Gly Gln 355 360 365 CAG TGCATG GAC CCT GTC AAA GGG GTC CTC TTC AAC GGA ATT ATC AAG 1152 Gln Cys MetAsp Pro Val Lys Gly Val Leu Phe Asn Gly Ile Ile Lys 370 375 380 GGT CCGGAT GGA CAA ATA TTG ATT CCA GAG ATG CAG TCA GAG CAG CTC 1200 Gly Pro AspGly Gln Ile Leu Ile Pro Glu Met Gln Ser Glu Gln Leu 385 390 395 400 AAACAG CAT ATG GAT CTG TTG AAA GCA GCT ATG TTT CCT CTC CGT CAT 1248 Lys GlnHis Met Asp Leu Leu Lys Ala Ala Met Phe Pro Leu Arg His 405 410 415 CCTTTA ATC AAC AGA GAG GCA GTC TTC AAG AAG GAT GGA AAT GCC GAT 1296 Pro LeuIle Asn Arg Glu Ala Val Phe Lys Lys Asp Gly Asn Ala Asp 420 425 430 GATTTT GTT GAT CTC CAT ATG CCT GAT GTT CAA AAA TCT GTG TCG GAT 1344 Asp PheVal Asp Leu His Met Pro Asp Val Gln Lys Ser Val Ser Asp 435 440 445 GTCGAC CTG GGC CTG CCT CAT TGG GGG TTC TGG TTG TTA GTC GGG GCA 1392 Val AspLeu Gly Leu Pro His Trp Gly Phe Trp Leu Leu Val Gly Ala 450 455 460 ACAGTA GTA GCC TTT GTG GTC TTG GCG TGC TTG CTC CGT GTA TGT TGT 1440 Thr ValVal Ala Phe Val Val Leu Ala Cys Leu Leu Arg Val Cys Cys 465 470 475 480AGG AGA ATG AGA AGG AGA AGG TCA CTG CGT GCC ACT CAG GAT ATC CCC 1488 ArgArg Met Arg Arg Arg Arg Ser Leu Arg Ala Thr Gln Asp Ile Pro 485 490 495CTC AGC GTT GCC CCT GCC CCT GTC CCT CGT GCC AAA GTG GTG TCA TCA 1536 LeuSer Val Ala Pro Ala Pro Val Pro Arg Ala Lys Val Val Ser Ser 500 505 510TGG GAG TCT TCT AAA GGG CTC CCA GGT ACT TGA 1569 Trp Glu Ser Ser Lys GlyLeu Pro Gly Thr 515 520 522 amino acids amino acid linear protein notprovided 10 Met Asn Ile Pro Cys Phe Ala Val Ile Leu Ser Leu Ala Thr ThrHis 1 5 10 15 Ser Leu Gly Glu Phe Pro Leu Tyr Thr Ile Pro Glu Lys IleGlu Lys 20 25 30 Trp Thr Pro Ile Asp Met Ile His Leu Ser Cys Pro Asn AsnMet Leu 35 40 45 Ser Glu Glu Glu Gly Cys Asn Thr Glu Ser Pro Phe Thr TyrPhe Glu 50 55 60 Leu Lys Ser Gly Tyr Leu Ala His Gln Lys Val Pro Gly PheThr Cys 65 70 75 80 Thr Gly Val Val Asn Glu Ala Glu Thr Tyr Thr Asn PheVal Gly Tyr 85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Lys Pro ThrVal Ala Ala 100 105 110 Cys Arg Asp Ala Tyr Asn Trp Lys Val Ser Gly AspPro Arg Tyr Glu 115 120 125 Glu Ser Leu His Thr Pro Tyr Pro Asp Ser SerTrp Leu Arg Thr Val 130 135 140 Thr Thr Thr Lys Glu Ala Leu Leu Ile IleSer Pro Ser Ile Val Glu 145 150 155 160 Met Asp Ile Tyr Gly Arg Thr LeuHis Ser Pro Met Phe Pro Ser Gly 165 170 175 Lys Cys Ser Lys Leu Tyr ProSer Val Pro Ser Cys Thr Thr Asn His 180 185 190 Asp Tyr Thr Leu Trp LeuPro Glu Asp Ser Ser Leu Ser Leu Ile Cys 195 200 205 Asp Ile Phe Thr SerSer Ser Gly Gln Lys Ala Met Asn Gly Ser Arg 210 215 220 Ile Cys Gly PheLys Asp Glu Arg Gly Phe Tyr Arg Ser Leu Lys Gly 225 230 235 240 Ser CysLys Leu Thr Leu Cys Gly Lys Pro Gly Ile Arg Leu Phe Asp 245 250 255 GlyThr Trp Val Ser Phe Thr Lys Pro Asp Val His Val Trp Cys Thr 260 265 270Pro Asn Gln Leu Val Asn Ile His Asn Asp Arg Leu Asp Glu Val Glu 275 280285 His Leu Ile Val Asp Asp Ile Ile Lys Lys Arg Glu Glu Cys Leu Asp 290295 300 Thr Leu Glu Thr Ile Leu Met Ser Gln Ser Val Ser Phe Arg Arg Leu305 310 315 320 Ser His Phe Arg Lys Leu Val Pro Gly Tyr Gly Lys Ala TyrThr Ile 325 330 335 Leu Asn Gly Ser Leu Met Glu Thr Asn Val Tyr Tyr LysArg Val Asp 340 345 350 Arg Trp Ala Asp Ile Leu Pro Ser Arg Gly Cys LeuLys Val Gly Gln 355 360 365 Gln Cys Met Asp Pro Val Lys Gly Val Leu PheAsn Gly Ile Ile Lys 370 375 380 Gly Pro Asp Gly Gln Ile Leu Ile Pro GluMet Gln Ser Glu Gln Leu 385 390 395 400 Lys Gln His Met Asp Leu Leu LysAla Ala Met Phe Pro Leu Arg His 405 410 415 Pro Leu Ile Asn Arg Glu AlaVal Phe Lys Lys Asp Gly Asn Ala Asp 420 425 430 Asp Phe Val Asp Leu HisMet Pro Asp Val Gln Lys Ser Val Ser Asp 435 440 445 Val Asp Leu Gly LeuPro His Trp Gly Phe Trp Leu Leu Val Gly Ala 450 455 460 Thr Val Val AlaPhe Val Val Leu Ala Cys Leu Leu Arg Val Cys Cys 465 470 475 480 Arg ArgMet Arg Arg Arg Arg Ser Leu Arg Ala Thr Gln Asp Ile Pro 485 490 495 LeuSer Val Ala Pro Ala Pro Val Pro Arg Ala Lys Val Val Ser Ser 500 505 510Trp Glu Ser Ser Lys Gly Leu Pro Gly Thr 515 520

What is claimed is:
 1. A method for expressing a protein of interest ina non-human mammal wherein said protein of interest is encoded by apolynucleotide contained within the genome of a recombinant retrovirus,and said polynucleotide is integrated into the genome of a non-humanmammalian unfertilized oocyte, comprising the steps of a) providing: i)an unfertilized non-human mammalian egg comprising an oocyte having aplasma membrane and a zona pellucida, said plasma membrane and said zonapellucida defining a perivitelline space; ii) an aqueous solutioncontaining said recombinant retrovirus, wherein said recombinantretrovirus comprises said polynucleotide encoding said protein ofinterest; b) introducing said solution containing said recombinantretrovirus into said perivitelline space under conditions which permitthe infection of said oocyte to provide an infected oocyte; c)contacting said infected oocyte with sperm under conditions which permitthe fertilization of said infected oocyte to produce an embryo; d)transferring said embryo into a hormonally synchronized non-humanmammalian recipient animal; e) allowing said embryo to develop into atleast one viable transgenic mammalian animal, under conditions such thatsaid protein of interest is expressed at detectable levels by saidtransgenic mammalian animal.
 2. The method of claim 1, wherein saidunfertilized oocyte is a pre-maturation oocyte.
 3. The method of claim2, further comprising following the introduction of said solutioncontaining retrovirus into said pre-maturation oocyte, the further stepof culturing said pre-maturation oocyte under conditions which permitthe maturation of said pre-maturation oocyte.
 4. The method of claim 1,wherein said unfertilized oocyte is a pre-fertilization oocyte.
 5. Themethod of claim 1, further comprising identifying at least onetransgenic non-human mammalian offspring.
 6. The method of claim 1,wherein said mammal is a bovine.
 7. The method of claim 1, wherein saidrecombinant retrovirus comprises Moloney murine leukemia virus longterminal repeat.